Antibody-drug conjugates

Abstract

The present invention relates to an antibody-drug conjugate targeting c-Kit and uses thereof.

Description

[Technical field]

[0001] The present invention relates to an antibody-drug conjugate targeting c-Kit and uses thereof. [Background technology]

[0002] Binding of stem cell factor (SCF) to c-Kit (also known as CD117) activates various signaling pathways including phosphoinositide 3-kinase, phospholipase C-gamma, Src kinase, and Janus kinase-signal transducer and activator of transcription (Yasuda A et al., Dig Dis Sci 52, 2292-2300. (2007); Sun J, Pedersen M & Ronnstrand L, J Biol Chem 284, 11039-11047. (2009)).

[0003] Previous studies have reported that expression of c-Kit harboring oncogenic mutations is unregulated or upregulated in various cancers, leading to SCF-independent c-Kit activation and cell proliferation. Overexpression of c-Kit is induced by hypoxia as well as activation of various transcription factors, including AP-2, ETS, SP1, MYB, and MITF. Currently, more than 500 c-Kit mutations have been identified in human tumors (Sanger Institute Catalog of Somatic Mutations in Cancer, https: / / cancer.sanger.ac.uk ). SCF-independent spontaneously activating c-Kit mutations are detected in approximately 85%, 30%, 25%, 25% and 90% of gastrointestinal stromal tumors (GIST), acute myeloid leukemia, acral melanoma, testicular tumors and systemic mastocytosis (SM) (Lennartsson J & Ronnstrand L, Physiol Rev 92, 1619-1649. (2012)).

[0004] The efficacy of small molecules such as imatinib to eliminate cancer stem cells harboring constitutively activated mutations of c-Kit is limited in tumor cells harboring mutations at different sites. To overcome c-Kit mutation-induced therapeutic resistance, various small molecules such as dasatinib, sunitinib, and axitinib have been developed (Abbaspour Babaei M et al., Drug Des Devel Ther 10, 2443-2459. (2016)). However, the rate of grade 3 / 4 abnormal reactions in patients treated with such small molecules is higher than that in patients treated with imatinib, limiting their effective application in cancer treatment (Kantarjian HM et al., Blood 119, 1123-1129. (2012)).

[0005] Antibody-drug conjugates (ADCs) enable the delivery of potent cytotoxic agents using the specificity of monoclonal antibodies to cancer cells. Recently, various ADCs targeting CD30 (brentuximab vedotin), CD22 (inotuzumab ozogamicin), CD79b (polatuzumab vedotin), and HER2 (trastuzumab emtansine and trastuzumab teruxtecan) have been approved by the US Food and Drug Administration (FDA) (Leung D et al., Antibodies (Basel) 9, 2. (2020)). Such ADCs are antibody-based drugs that bind to the extracellular domain of the target molecule, and therefore have the relative advantage of being free of mutations that mainly occur in the intracellular domain and can be applied regardless of various mutations. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an antibody-drug conjugate of the formula: Ab-(L1) m -(S) n -(L2) p -(D) q In the above formula, Ab is an anti-c-Kit antibody or antigen-binding fragment thereof that specifically binds to the epitopes of human c-Kit in SEQ ID NO:9 and SEQ ID NO:10; L1 is a linker connecting the Ab and S or the Ab and L2; S is a spacer with or without a polymer attached; L2 is a cleavable linker; D is a drug moiety; m is an integer from 0 to 8; n is an integer from 0 to 8; p is an integer from 1 to 8; q is an integer from 1 to 8.

[0007] Another object of the present invention is to provide an antibody-drug conjugate of the formula: Ab-(L) x -(D) y In the above formula, Ab is an anti-c-Kit antibody or antigen-binding fragment thereof that specifically binds to the epitopes of human c-Kit in SEQ ID NO:9 and SEQ ID NO:10; L is a linker, including a cleavable linker; D is a drug moiety; x is an integer from 1 to 8; and y is an integer from 1 to 8.

[0008] It is yet another object of the present invention to provide a composition comprising the antibody-drug conjugate.

[0009] It is yet another object of the present invention to provide a method for preventing or treating cancer, comprising administering a pharma- ceutical effective amount of the composition to a subject in need thereof.

[0010] It is yet another object of the present invention to provide a method for the use of said composition in therapy.

[0011] It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, comprising the antibody-drug conjugate.

[0012] It is still another object of the present invention to provide a use for the production of said pharmaceutical composition.

[0013] It is still another object of the present invention to provide a composition for diagnosing cancer, comprising the antibody-drug conjugate.

[0014] It is yet another object of the present invention to provide a method for providing information for diagnosing cancer, comprising the step of treating a sample separated from a subject with the antibody-drug conjugate.

[0015] Yet another object of the present invention is to provide an antibody or antigen-binding fragment (Ab) that specifically binds to human c-Kit (L1). m -(S) n -(L2) p -(D) q The present invention provides a method for producing an anti-c-Kit antibody-drug conjugate, comprising the step of conjugating In the above formula, L1 is a linker connecting the Ab and S or the Ab and L2; S is a spacer with or without a polymer attached; L2 is a cleavable linker; D is a drug moiety; m is an integer from 0 to 8; n is an integer from 0 to 8; p is an integer from 1 to 8; q is an integer from 1 to 8.

[0016] It is yet another object of the present invention to provide a method for producing an anti-c-Kit antibody-drug conjugate, comprising the step of conjugating a linker (L)-drug (D) to an antibody or an antigen-binding fragment thereof comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6, wherein L is a linker comprising a cleavable linker, and D is a drug moiety.

[0017] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0018] The present invention relates to an antibody-drug conjugate and its use, and demonstrates that cancer can be controlled, treated, alleviated, or reduced, or recurrence can be suppressed, for example, by administration of an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof that specifically binds to human c-Kit.

[0019] To facilitate the understanding of this invention, a number of terms and phrases are defined. Further definitions are presented throughout the detailed description.

[0020] I. Definition The term "about" is used herein to mean approximately, roughly, around, or within the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value above and below the stated value, for example, up or down (higher or lower), up to a variance of 10%.

[0021] Also, "and / or" should be construed as specifically disclosing each of the two specified features or components in conjunction with or alone the remaining one. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to include the embodiments A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single), respectively.

[0022] As used herein, the terms "treat," "treat" and "treatment" refer to any type of intervention or process performed on a subject or the administration of an active agent to a subject for the purpose of reversing, ameliorating, alleviating, inhibiting or delaying the progression, occurrence, severity or recurrence of symptoms, complications, conditions or biochemical indicators associated with a disease. Treatment can be performed on subjects with a disease or subjects not having a disease (e.g., prophylaxis).

[0023] The term "administration" as used herein refers to the physical introduction of a therapeutic agent or a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Different routes of administration for the antibody-drug conjugates disclosed herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. The term "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, generally by injection, including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intraspinal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intraorgan, pulmonary, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and substernal injection and infusion, as well as in vivo electroporation. Alternatively, the antibody drug conjugates disclosed herein may be administered via a parenteral route, e.g., via a topical, epithelial, or mucosal administration route, e.g., via intranasal, oral, vaginal, rectal, sublingual, or local route. Administration may also be performed, e.g., once, multiple times, and / or over one or more extended periods of time.

[0024] As used herein, the term "therapeutically effective amount" refers to an amount of a drug, alone or in combination with other therapeutic agents, that is effective to "treat" a disease or disorder in a subject or to reduce the risk, latency, likelihood or occurrence of a disease or disorder (e.g., cancer). A "therapeutically effective amount" includes an amount of a drug or therapeutic agent that provides some improvement or benefit to a subject having or at risk of having a disease or disorder (e.g., cancer). Thus, a "therapeutically effective amount" is an amount that reduces the risk, latency, likelihood or occurrence of a disease or disorder, or provides some relief, alleviation, and / or reduces at least one indicator (e.g., cancer) and / or reduces at least one clinical symptom of the disease or disorder.

[0025] The term "cancer" as used herein refers to a broad group of diverse diseases characterized by uncontrolled growth of abnormal cells in the body. "Cancer" or "cancerous tissue" may include tumors. Dysregulated cell division and growth leads to the formation of malignant tumors that invade neighboring tissues, which may also metastasize to distant locations in the body via the lymphatic system and bloodstream. After metastasis, distal tumors may be said to have "derived from" the pre-metastatic tumor. For example, a "tumor derived from" a melanoma refers to a tumor that is the result of a melanoma that has metastasized. Since a distal tumor originates from a pre-metastatic tumor, "derived from" a tumor may also include a pre-metastatic tumor, for example, a tumor derived from a melanoma may include a melanoma.

[0026] As used herein, the phrase "inhibiting tumor growth" includes any measurable reduction in tumor growth, for example, inhibition of tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or up to 100%.

[0027] The term "effective amount", "pharmaceutical effective amount" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is an amount of the drug that, when used alone or in combination with other therapeutic agents, promotes disease regression evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disease impairment or injury. A therapeutically effective amount or effective dosage of a drug includes a "prophylactically effective amount" or "prophylactically effective dosage," which is an amount of a drug that inhibits disease onset or recurrence when administered alone or in combination with other therapeutic agents to a subject at risk of developing a disease or suffering from a recurrence of the disease. The efficacy of a therapeutic agent to promote disease regression or inhibit disease onset or recurrence can be evaluated using a variety of methods known to those skilled in the art, for example, by analyzing the activity of the formulation in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0028] By way of example, an anti-cancer drug is a drug that promotes cancer regression in a subject. In some embodiments, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administration of a pharmacologic effective amount of the drug, alone or in combination with an anti-neoplastic drug, results in a decrease in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease symptom-free periods, prevention of disease injury or damage, or other improvement of disease symptoms in a patient. In addition, the terms "effective" and "efficacy" encompass both pharmacological efficacy and physiological safety in the context of treatment. Pharmacological efficacy refers to the efficacy of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other negative physiological effects (side effects) at the cellular, organ, and / or organismal level that result from administration of the drug.

[0029] For example, in the case of tumor treatment, a therapeutically effective amount or effective dosage of a drug inhibits cell or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% compared to untreated subjects. In some embodiments, a therapeutically effective amount or effective dosage of a drug completely inhibits cell or tumor growth, i.e. inhibits cell or tumor growth by 100%. The ability of an inhibitor to inhibit tumor growth can be evaluated using the assays described below. Alternatively, this property of the composition can be evaluated by testing the ability of the inhibitor to inhibit cell growth, such inhibition can be measured in vitro by assays known to those skilled in the art. In other embodiments disclosed herein, tumor regression can be observed and can last for at least about 20 days, at least about 40 days, or at least about 60 days.

[0030] Some aspects of the invention relate to diagnosing cancer in a subject.

[0031] The term "diagnosis" as used herein refers to a method that can be used to determine or predict whether a patient is afflicted with a given disease or condition. Those skilled in the art can make a diagnosis based on one or more diagnostic markers (e.g., expression level of c-Kit), where the presence, absence, amount, or change in amount of the diagnostic marker indicates the presence, severity, or absence of the condition. In some embodiments, increased expression of c-Kit in a biological sample from a subject is indicative for a tumor. The term "diagnosis" does not refer to the ability to determine the presence or absence of a particular disease with 100% accuracy, nor does it mean that a given course or outcome is more likely to occur than not. Instead, those skilled in the art will understand that the term "diagnosis" refers to an increased probability that a particular disease exists in a subject.

[0032] The term "diagnostic marker" (e.g., c-Kit expression) refers to a substance capable of distinguishing and diagnosing tumors from normal cells, and includes organic biological molecules, such as polypeptides, or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, and sugars (monosaccharides, disaccharides, oligosaccharides, etc.), which are increased or decreased in tumor cells. The diagnostic markers disclosed herein for cancer can be a protein expressed from the c-Kit gene, whose expression is increased in tumor cells.

[0033] Compositions for diagnosing cancer include preparations for measuring the expression level of mRNA of the c-Kit gene or the amount of expressed protein. Such preparations include an oligonucleotide having a sequence complementary to c-Kit mRNA, a primer or a nucleic acid probe that specifically binds to c-Kit mRNA, and an antibody or antigen-binding fragment thereof that specifically binds to c-Kit protein (hereinafter, anti-c-Kit antibody), or an antibody-drug conjugate in which the anti-c-Kit antibody is linked to a drug via a linker.

[0034] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0035] The term "c-Kit" belongs to class III receptor tyrosine kinase (RTK) and is also known as the receptor for SCF.

[0036] "c-Kit" includes any variant or isoform of c-Kit naturally expressed by cells. Thus, the anti-c-Kit antibodies or antibody-drug conjugates disclosed herein may cross-react with different isoforms of the same species (e.g., different isoforms of human c-Kit) or may cross-react with c-Kit of a species other than human (e.g., mouse c-Kit). Alternatively, the anti-c-Kit antibodies may be specific for human c-Kit and may not show cross-reactivity with other species. c-Kit, or any variants and isoforms thereof, may be isolated from cells or tissues that naturally express them or produced by recombinant technology. The sequence of domains 1 to 3 of human c-Kit, excluding the 25-mer signal peptide (Q26 to D309), may be SEQ ID NO:12.

[0037] The term "antibody" is a term of the art and may be used interchangeably herein to refer to a molecule having an antigen-binding site that specifically binds to an antigen. As used herein, the term includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. In one embodiment, "antibody" refers to a glycoprotein comprising at least two heavy (H) and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. In another embodiment, "antibody" refers to a single-chain antibody comprising a single variable domain, e.g., a VHH domain. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region is composed of three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL.

[0038] The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the canonical complement system.

[0039] The antibody can have any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgD, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subtype (e.g., IgG1, IgG2, IgG3 and IgG4 in humans; and IgG1, IgG2a, IgG2b and IgG3 in mice) of immunoglobulin molecule. Immunoglobulins, e.g., IgG1, exist in several allotypes, which differ from each other by up to a few amino acids. The antibodies disclosed herein can be derived from any of the commonly known isotypes, classes, subtypes, or allotypes. In certain embodiments, the antibodies disclosed herein are of the IgG1, IgG2, IgG3, or IgG4 subtypes, or any hybrids thereof. In certain embodiments, the antibodies are of the human IgG1 subtype or human IgG2 or human IgG4 subtype.

[0040] "Antibody" includes, by way of example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies, totally synthetic antibodies; single chain antibodies; monospecific antibodies; multispecific antibodies (including bispecific antibodies); tetrameric antibodies comprising two heavy chain and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers; antibody heavy chain dimers; antibody light chain-antibody heavy chain pairs; intrabodies; heteroconjugate antibodies; monovalent antibodies; camelid antibodies; apibodies; anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies), and single domain antibodies (sdAbs), which include binding molecules comprised of a single monomeric variable antibody domain (e.g., a VH domain or a VL domain) sufficient for antigen binding (Harmen MM and Haard HJ Appl Microbiol Biotechnol. 77(1):13-22 (2007)).

[0041] As used herein, the term "antigen-binding portion" of an antibody, i.e., "antigen-binding fragment", refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., domain 2 / 3 of human c-Kit). Such a "fragment" is, for example, about 8 to about 1500 amino acids in length, preferably about 8 to about 745 amino acids in length, more preferably about 8 to about 300, e.g., about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been revealed that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody, e.g., an anti-c-Kit antibody disclosed herein, include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment composed of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment composed of the VH and CH1 domains; (iv) an Fv fragment composed of the VL and VH domains of a single arm of an antibody, and a disulfide-linked Fvs (sdFv); (v) a dAb fragment composed of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) a separate complementarity determining region (CDR) or (vii) a combination of two or more separate CDRs which can be optionally linked by a synthetic linker. Also, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be linked using recombinant methods by a synthetic linker, so that the VL and VH regions pair to form a single protein chain (referred to as single-chain Fv (scFv)) (see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also included within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0042] As used herein, the terms "variable region" or "variable domain" are commonly used interchangeably in the art. A variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically about the amino terminal 110-120 amino acids in the mature heavy chain and about 90-115 amino acids in the mature light chain, which differ extensively in sequence from antibodies and are used due to the binding and specificity of a particular antibody for its particular antigen. Sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions of the variable domain are called framework regions (FRs).

[0043] The CDRs of the light and heavy chains are considered to be primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and a human framework region (FR). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and a primate (e.g., non-human primate) framework region (FR).

[0044] As used herein, the term "heavy chain", when used in connection with an antibody, refers to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the constant domain, which give rise to the IgA, IgD, IgE, IgG and IgM types of antibodies, including subtypes of IgG, e.g., IgG1, IgG2, IgG3 and IgG4, respectively.

[0045] As used herein, the term "light chain" when used in connection with an antibody refers to any of the different types, such as capacitive (κ) and lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0046] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0047] The terms "VH" and "VH domain" are used interchangeably to refer to the variable region of an antibody heavy chain.

[0048] The terms "constant region" or "constant domain" as used herein are interchangeable and have their usual meaning in the art. A constant domain is a portion of an antibody, e.g., the carboxy-terminal portion of the light and / or heavy chain, that is not directly involved in binding an antibody to an antigen, but can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain.

[0049] "Fc region" (fragment determinable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or the first component of the canonical complement system (C1q). Thus, the Fc region includes the constant regions of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA and IgD antibody isotypes, the Fc region includes two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains; IgM and IgE Fc regions include three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. In the case of IgG, the Fc region includes immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position C226 or P230 (or an amino acid between these two amino acids) to the carboxy-terminus of the heavy chain, where numbering is according to the EU index as in Kabat. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, and the CH3 domain is located C-terminal to the Cm domain in the Fc region, i.e., it extends from about amino acid 341 to about amino acid 447 of IgG. The Fc region may be a native sequence Fc, including any allotypic variants, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may refer to the region in isolation or in association with an Fc-containing protein polypeptide, such as an "Fc region-containing binding protein," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesion).

[0050] "Hinge", "hinge domain" or "hinge region" or "antibody hinge region" refers to the domain of the heavy chain constant region that connects the CH1 domain to the CH2 domain and includes the upper, middle, and lower portions of the hinge (Roux et al. J. Immunol. 1998 161:4083). The hinge provides varying levels of flexibility between the binding and effector regions of an antibody and also provides a site for intermolecular disulfide bonds between the two heavy chain constant regions. The sequences of wild-type IgG1, IgG2, IgG3 and IgG4 hinges are known in the art (see, e.g., Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al., Front Immunol. 5:520 (Published online October 20, 2014)).

[0051] As used herein, "isotype" refers to the antibody type (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by heavy chain constant region genes.

[0052] "Allotype" refers to naturally occurring variants within a particular isotype group that differ by some amino acids (see, for example, Jefferis et al. (2009) mAbs 1:1). The antibodies presented herein can have any allotype. The allotypes of IgG1, IgG2, IgG3, and IgG4 are known in the art (see, for example, Kabat EA et al., (1991) (homologous); Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014); and Lefranc MP, mAbs 1:4,1-7 (2009)).

[0053] The terms "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0054] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds c-Kit is substantially free of antibodies that specifically bind to other antigens other than c-Kit). However, an isolated antibody that specifically binds to an epitope of c-Kit may have cross-reactivity to other c-Kit proteins from different species.

[0055] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for a partner Y can generally be expressed by a dissociation constant (KD). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD) and equilibrium binding constant (KA). KD is calculated from the quotient of koff / kon and expressed in molar concentration (M), and KA is calculated from the quotient of kon / koff. kon refers to, for example, the binding rate constant of an antibody to an antigen, and koff refers to, for example, the dissociation rate constant of an antibody to an antigen. Kon and koff can be determined by techniques known to those of skill in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), BIACORE®, or dynamic exclusion assays (KinExA®).

[0056] As used herein, the terms "specifically bind," "specifically recognize," "specific binding," "selective binding," and "selectively bind" are similar terms in the context of antibodies and molecules (e.g., antibodies) that bind to an antigen (e.g., an epitope or immune complex), as such binding would be understood by one of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, a BIACORE®, KinExA® 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art.

[0057] Antibodies are typically -5 ~10 -11 It specifically binds to its cognate antigen with high affinity, as reflected by a dissociation constant of approximately 10 -4 A KD greater than M is generally considered to indicate non-specific binding. An antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen with high affinity and substantially the same antigen, for example, as determined by immunoassay (e.g., ELISA) or surface plasmon resonance (SPR) technology on a BIACORE® 2000 instrument using a defined antigen, with a KD of 10 -7 M or less, preferably 10 -8 M or less, more preferably 10 -9 M or less, more preferably 10 -10 M or less or 10 -11 M or less, which means that it does not bind with high affinity to unrelated antigens.

[0058] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. The antigen may be c-Kit or a fragment thereof.

[0059] "Epitope" as used herein is a term of the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, a polypeptide or a combination of two or more non-contiguous regions of a polypeptide (conformational, non-linear, discontinuous, or discontinuous epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained upon exposure to denaturing solvents, and epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation analysis, in which overlapping or consecutive peptides (e.g., c-Kit domains 2 and 3) are tested for reactivity with a given antibody (e.g., anti-c-Kit antibody). Methods for determining the spatial conformation of epitopes include those known in the art and disclosed herein, including, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0060] The term "binds to the same epitope" in the context of two or more antibodies means that the antibodies bind to the same segment of amino acid residues as determined by a given method. Techniques for determining whether an antibody binds to the "same epitope on c-Kit" as the antibodies presented herein include, for example, epitope mapping methods, such as X-ray analysis of crystals of antigen:antibody complexes and hydrogen / deuterium exchange mass spectroscopy (HDX-MS), which provide atomic resolution of the epitope. Other methods monitor the binding of antibodies to antigen fragments or mutated variants of the antigen, where loss of binding due to deformation of amino acid residues within the antigen sequence is primarily considered as an indication of the epitope component. In addition to this, combinatorial computational methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from a combinatorial phage display peptide library. Antibodies with the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

[0061] An antibody that "cross-competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the remaining antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to the target, can be determined using known competition experiments. In certain embodiments, an antibody competes with another antibody for binding to a target and inhibits this binding by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition differs depending on whether the antibody is a "blocking antibody" (i.e., a cold antibody that has been previously incubated with the target). Competitive analysis can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999). Competitive antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., by steric hindrance).

[0062] The term "reference antibody" refers to an antibody that serves as a standard for analysis of competing antibodies that bind to the same epitope, an overlapping epitope, or an adjacent epitope, and cross-competes with the competing antibodies in binding to the epitope.

[0063] Other competitive binding assays include solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990); and direct labeling RIA (see Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0064] c-Kit is composed of five extracellular domains composed of IgG-like repeats, a cytoplasmic kinase domain containing ATP-binding (TK1) and phosphotransferase (TK2) domains separated by juxtamembrane and kinase inserts. Upon ligand binding, c-Kit is activated by phosphorylation via ligand-mediated dimerization. Consequently, signaling mediators including Src, PI3K, STAT1 and JAK2 are recruited to regulate diverse cell-specific responses such as proliferation, survival and motility. Overexpression of RTKs is also known to spontaneously activate them to induce cell growth in a ligand-independent manner. Overexpression and activation of c-Kit have been reported in a variety of cancers including GBM, astrocytoma, germ cell and SCLC and correlate with poor prognosis.

[0065] The 2G4 antibody is a fully human anti-c-Kit antibody that binds to domains 2 / 3 of c-Kit and blocks SCF binding, thereby inhibiting c-Kit. The specific c-Kit binding site of 2G4 has been confirmed using a 2G4 / c-Kit complex docking model (J.-O. Kim et al., International Journal of Biological Macromolecules 159(2020)66-78), which is incorporated herein by reference.

[0066] To understand the molecular mechanism of the 2G4 antibody, molecular docking analysis was performed against the crystal structure of 2G4 Fab and c-Kit (PDB ID: 2E9W) using the HADDOCK / ZDOCK program. Residues 26-309 (D1-D3 region, SEQ ID NO: 12) of c-Kit were used for the docking calculation as follows:

[0067] First, blind docking calculations were performed using ZDOCK. The 10 model structures with the highest ZDOCK scores generally indicate that the CDR region of 2G4 Fab interacts with the D2 and D3 regions of c-Kit (D113-D309; SEQ ID NO: 11). Based on the model structure of the 2G4 Fab:c-Kit complex, five residues (R122, Y125, R181, K203 and K205) were selected from the c-Kit D2 region (SEQ ID NO: 9), and five residues (S240, S241, Y243, N260 and W262) were selected from the c-Kit D3 region (SEQ ID NO: 10). The selected residues were expected to be responsible for 2G4 binding. Next, 10 mutant c-Kit proteins were generated, and the binding affinity of 2G4 was compared with the wild-type and mutant c-Kit proteins using ELISA (Figures 14a to 14c). ELISA results indicated that residues R122, Y125, R181, K203, K205, S261 and H263 of c-Kit were important for binding to 2G4.

[0068] The second docking calculation was performed using HADDOCK. The key residues of c-Kit identified by mutagenesis and ELISA analysis and all residues constituting the CDR of 2G4 were used as constraints. The docking model calculated by HADDOCK showed that the relative orientation and binding interface between 2G4 and c-Kit (i.e., the CDR loops of 2G4 and the D2 / D3 region of c-Kit) were similar to those calculated by ZDOCK. We confirmed that the heavy chain of 2G4 and the D2-D3 region of c-Kit are closely related in the structural model of the 2G4 Fab:c-Kit complex with the highest score (Figure 14d). Specifically, the binding interface between 2G4 Fab and c-Kit is mainly formed by electrostatic interactions between the heavy chain CDR of 2G4 and the D2 region of c-Kit. Basic amino acids of c-Kit, including R122, R181, K203 and R205, were shown to interact with negatively charged residues of 2G4 (D74H, E119H, D120H, E123H and D126H) (Fig. S14e). Additional interactions between S261 and H263 in the D3 region of c-Kit and light chain residues of 2G4 (D82L, Q118L, T119L) also contributed to the formation of the complex (Fig. S14e).

[0069] Through the docking calculation, it was demonstrated that 2G4 binds to R122, Y125, R181, K203, R205, S261 and H263 in the D2 / 3 region of c-Kit.

[0070] Thus, the present invention provides an antibody-drug conjugate, wherein the antibody or antigen-binding fragment thereof specifically binds to c-Kit. In a particular embodiment of the antibody-drug conjugate, the antibody or antigen-binding fragment thereof specifically binds to epitopes in domains 1-3 of human c-Kit (SEQ ID NO: 12), more specifically, domains 2 / 3 of human c-Kit (SEQ ID NO: 11), more specifically, the R122-R205 region of domain 2 of human c-Kit (SEQ ID NO: 9) and the S240-H263 region of domain 3 of human c-Kit (SEQ ID NO: 10), most specifically, the R122, Y125, R181, K203, R205, S261, and H263 epitopes in domains 2 / 3 of human c-Kit.

[0071] The term "monoclonal antibody" as used herein refers to an antibody or a composition of antibodies in which all of the antibodies exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody or antibody composition that exhibits a single binding specificity and has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, a human monoclonal antibody is produced by a hybridoma that includes B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse whose genome includes human heavy and light chain transgenes fused to an immortalized cell.

[0072] The term "recombinant human antibody" includes all human antibodies that are produced, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes, or hybridomas produced therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, (c) antibodies isolated from human combinatorial antibody libraries, and (d) antibodies produced, expressed, produced or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that are encoded by germline genes but use specific human germline immunoglobulin sequences including subsequent rearrangements and mutations that occur, e.g., during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable region contains the antigen-binding domains encoded by the various genes that rearrange to form antibodies specific to foreign antigens. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (somatic mutation or hypermutation) to increase the affinity of the antibody to the foreign antigen. The constant region changes in further response to the antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid molecules that encode light and heavy chain immunoglobulin polypeptides in response to an antigen cannot have sequence identity to the original nucleic acid molecule, but instead are substantially identical or similar (i.e., have at least 80% identity).

[0073] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Also, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The antibodies presented herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mouse, are joined to human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0074] The term "humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDR domain of a non-human antibody are replaced with the corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domain are replaced with amino acids from a human immunoglobulin, and some, most or all of the amino acids in one or more CDR regions remain intact. Amino acid additions, deletions, insertions, substitutions or modifications are permissible as long as they do not eliminate the ability of the antibody to bind to a particular antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0075] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, e.g., the variable region is derived from a murine antibody and the constant region is derived from a human antibody.

[0076] The term "cross-react" as used herein refers to the ability of the antibody presented herein to bind to c-Kit from different species. For example, an antibody presented herein that binds to human c-Kit can also bind to c-Kit from other species (e.g., mouse c-Kit). Such cross-reactivity can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA), or by detecting binding or functional interaction with cells that physiologically express c-Kit. Methods for determining cross-reactivity include standard binding assays disclosed herein, such as BIACORE® surface plasmon resonance (SPR) assays using a BIACORE® 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometry.

[0077] The term "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, predicted non-essential amino acid residues in anti-c-Kit antibodies are replaced with other amino acid residues from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not impair antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0078] The term "substantial homology" indicates that two polypeptides, or their designated sequences, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80% of the amino acids, at least about 90%-95%, or at least about 98%-99.5% of the amino acids.

[0079] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that should be introduced for optimal alignment of the two sequences (i.e., % homology = # of identical positions / # of positions x 100). Comparison of sequences and percent identity between two sequences can be determined using a mathematical algorithm, such as those described in the non-limiting examples below.

[0080] The percent identity between two amino acid sequences may be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm as integrated into the GAP program of the GCG software package (available at http: / / www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0081] The nucleic acid and protein sequences presented herein can be used as "query sequences" to perform searches in public databases, for example to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules presented herein. BLAST protein searches can be performed using the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to the protein molecules presented herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used (see worldwideweb.ncbi.nlm.nih.gov).

[0082] The term "linked" as used herein refers to the association of two or more molecules. Linkage can be covalent or non-covalent. Linkage can also be genetic (i.e., fused by recombinant techniques) linkage. Such linkage can be accomplished using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0083] The term "antibody-drug conjugate" as used herein refers to the linkage of an antibody or its antigen-binding fragment with further other agents, such as anti-cancer agents, chemotherapeutic agents, toxins, immunotherapeutic agents, imaging probes, spectroscopic probes, etc. Such linkages can be covalent or non-covalent interactions, such as interactions via electrostatic forces. A variety of linkers known in the relevant art can be used to form antibody-drug conjugates. Furthermore, antibody-drug conjugates can be provided in the form of fusion proteins that can be expressed from polynucleotides encoding the antibody-drug conjugates. As used herein, "fusion protein" refers to a protein generated through the linkage of two or more genes or gene fragments that originally encoded separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each of the original proteins.

[0084] As used herein, the term "spacer" refers to a substance that serves to prevent aggregation of an antibody-drug conjugate in the bloodstream and to impart stability in the bloodstream and targeted pharmacokinetic properties.

[0085] The term "linker" as used herein refers to any chemical moiety that can further link an antibody, antibody fragment (e.g., antigen-binding fragment) or functional equivalent to another moiety, e.g., a drug moiety. The linker can be susceptible to cleavage, e.g., acid-induced cleavage, photoinduced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, provided that the compound or antibody remains active (cleavable linker). Alternatively, the linker can be substantially resistant to cleavage (e.g., stable linker or non-cleavable linker). In some embodiments, the linker is a precharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0086] A "non-cleavable linker" has relatively high plasma stability and is resistant to proteolysis. When an antibody is degraded after being introduced into a cell, the drug is released in the form of a complex with the linker, and this complex has drug activity. A thioether linker is a representative non-cleavable linker, which allows the drug to be released after the antibody is non-selectively degraded in the cell. Kadcyla (registered trademark) adopts a non-cleavable method.

[0087] The term "cleavable linker" refers to a linker that can be cleaved in response to a specific environmental factor to release a drug into the cytoplasm. The cleavable type can be an enzyme-cleavable type or a non-enzyme-cleavable type.

[0088] The enzyme-cleaved form is cleaved by enzymes such as cathepsin B, β-glucuronidase, phosphatase, hydrophosphatase, and sulfatase. The peptide linker is degraded by protease, and protease inhibitors are present in plasma, resulting in high plasma stability. Cathepsin B is a typical protease used in antibody-drug conjugates, and cathepsin B is present at high levels in tumor tissues, conferring tumor selectivity to antibody-drug conjugates. The peptide linker can be a dipeptide consisting of two amino acids linked together. The β-glucuronide linker is degraded by β-glucuronidase, a glycolytic enzyme present in lysosomes. β-glucuronidase is overexpressed in some tumor cells, conferring tumor specificity.

[0089] Non-enzymatically cleavable linkers include acid-labile linkers and oxidation-reduction reaction linkers.

[0090] Acid-labile linkers are relatively less stable linkers that were developed early on, but are still in use. A typical example is the hydrazone linker, which is hydrolyzed in the weakly acidic environment of endosomes and lysosomes after internalization into tumor cells, releasing the drug.

[0091] A typical redox-reactive linker is a disulfide linker. After internalization, the linker is degraded by disulfide exchange or a reducing agent such as glutathione, releasing the cytotoxic drug. Glutathione is present in hypoxic tumor cells at concentrations approximately 1,000-fold higher than in normal cells, conferring tumor cell selectivity to ADCs.

[0092] Procharged linkers are derived from charged cross-linking reagents that maintain their charge after incorporation into an antibody drug conjugate. Examples of procharged linkers can be found in US 2009 / 0274713.

[0093] II. Antibody-Drug Conjugates (ADCs) According to one aspect of the present invention, there is provided an antibody-drug conjugate having the following formula 1: <Chemical formula 1> Ab-(L) x -(D) y In the above formula, Ab is an anti-c-Kit antibody or antigen-binding fragment thereof that specifically binds to the epitopes of human c-Kit in SEQ ID NO:9 and SEQ ID NO:10; L is a linker, including a cleavable linker; D is a drug moiety; x is an integer from 1 to 8; and y is an integer from 1 to 8.

[0094] In some specific examples, the Ab is an antibody or an antigen-binding fragment thereof that specifically binds to one or more of R122, Y125, R181, K203, R205, S261, and H263 in SEQ ID NO: 12. Preferably, the Ab is an antibody or an antigen-binding fragment thereof that specifically binds to R122, Y125, R181, K203, R205, S261, and H263 in SEQ ID NO: 12. In this case, SEQ ID NO: 12 is the sequence of domains 1 to 3 of human c-Kit (Q26 to D309) excluding 25 signal peptides, and the numbers of each amino acid are numbers including the 25 signal peptides. Thus, for example, the R122 means the 97th amino acid sequence in SEQ ID NO: 12, and the Y125 means the 100th amino acid sequence, and the remaining amino acids can be analyzed in the same manner.

[0095] In some embodiments, the antibody or antigen-binding fragment thereof cross-competes with a reference antibody comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6 for binding to the epitope of human c-Kit at SEQ ID NO: 9 and SEQ ID NO: 10.

[0096] In some embodiments, the antibody or antigen-binding fragment thereof cross-competes with a reference antibody comprising a heavy chain variable domain set forth in SEQ ID NO:7 and a light chain variable domain set forth in SEQ ID NO:8 for binding to the epitopes of human c-Kit at SEQ ID NO:9 and SEQ ID NO:10.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain represented by SEQ ID NO:7 and a light chain variable domain represented by SEQ ID NO:8.

[0099] The antibody or antigen-binding fragment thereof may, but is not limited to, have one or two amino acids in the CDRs modified, deleted or substituted.

[0100] In some embodiments, the antibodies or antigen-binding fragments thereof may maintain greater than 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity across the heavy chain variable or light chain heavy chain regions.

[0101] In some embodiments, the antibody or antigen-binding fragment thereof may be a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a human antibody, a single chain antibody (scFv) or an antibody fragment.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof has an equilibrium dissociation constant KD value for human c-KIT of 10 -11 M or less, specifically, for example, about 2.8237 (±0.9)X10 -12 It may be of M.

[0103] The cleavable linker includes, but is not limited to, a linker selected from the group consisting of valine-citrulline-p-aminobenzylcarbamoyl (Val-Cit-PAB), alanine-phenylalanine-p-aminobenzylcarbamoyl (Ala-Phe-PAB), alanine-alanine-p-aminobenzylcarbamoyl (Ala-Ala-PAB), valine-alanine-p-aminobenzylcarbamoyl (Val-Ala-PAB), phenylalanine-lysine-p-aminobenzylcarbamoyl (Phe-Lys-PAB), alanine-alanine-glycine-p-aminobenzylcarbamoyl (Ala-Ala-Gly-PAB), and glycine-glycine-glycine-p-aminobenzylcarbamoyl (Gly-Gly-Gly-PAB) linkers.

[0104] The L may include a spacer in the form of a polymer-bound or unbound spacer.

[0105] According to one embodiment of the present invention, the L is:

[0106] TIFF2025508737000002.tif62170

[0107] In one embodiment, the linker is a linker derived from a cross-linking reagent comprising a substituent selected from the group consisting of:

[0108] TIFF2025508737000003.tif36170

[0109] For example, the linker may include a disulfide bridge linking two cysteine ​​residues, such as in ThioBridge® (Abzena) technology, when prepared using a cross-linking reagent containing a substituent.

[0110] In the above chemical formula, the wavy line

[0111] TIFF2025508737000004.tif9170

[0112] indicates the site where another linker, S or D is attached.

[0113] The R can be hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, cyano, nitro, phenyl, toluene, or halophenyl.

[0114] In some embodiments, L is a linker that includes a spacer with or without a polymer attached.

[0115] In some embodiments, L comprises the structure of Formula 2: <Chemical formula 2> (L1) m -(S) n -(L2) p In the above formula, L1 is a linker connecting the Ab and S or the Ab and L2; S is a spacer with or without a polymer attached; L2 is a cleavable linker; m is an integer from 0 to 8; n is an integer from 0 to 8; and p is an integer from 1 to 8.

[0116] In some specific examples, said m is 1-4, said n is 1-4, and said p is 1-4.

[0117] In some embodiments, L1 comprises a linker selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a procharged linker, and a dicarboxylic acid-based linker.

[0118] In some embodiments, L or L1 is

[0119] TIFF2025508737000005.tif196170

[0120] TIFF2025508737000006.tif61170

[0121] Includes.

[0122] In the above chemical formula, the wavy line

[0123] TIFF2025508737000007.tif8170

[0124] indicates the site where Ab, or S, L2 or D binds.

[0125] In some embodiments, L1 is

[0126] TIFF2025508737000008.tif60170

[0127] and wherein the linker is derived from a cross-linking reagent that includes a substituent selected from the group consisting of:

[0128] In the above chemical formula, the wavy line

[0129] TIFF2025508737000009.tif7170

[0130] indicates the site where S binds.

[0131] The R can be hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, cyano, nitro, phenyl, toluene, or halophenyl.

[0132] According to one embodiment of the present invention, the S (spacer) is

[0133] TIFF2025508737000010.tif62170

[0134] may be selected from the group consisting of:

[0135] In the above chemical formula, the wavy line

[0136] TIFF2025508737000011.tif8170

[0137] indicates the site to which L1 or L2 binds, and preferably, L1 binds to the left wavy line side, and L2 binds to the right wavy line side.

[0138] In some embodiments, S can be a spacer comprising an aspartate, a glutamate, or a combination thereof, with or without a polymer attached.

[0139] Examples of the polymer include, but are not limited to, polyalkylene, polyalkylene glycol, polyvinylpyrrolidone, polyacrylate, polyoxazoline, polyvinylalcohol, polyacrylamide or polymethacrylamide, HPMA copolymer, polyester, polyacetal, poly(orthoester), polycarbonate, poly(iminocarbonate), polyamide, divinyl ether-maleic anhydride or styrene-maleic anhydride copolymer, polysaccharide, or polyglutamic acid.

[0140] In some embodiments, the polymer is polyethylene or polyethylene glycol.

[0141] The polyethylene glycol may contain 10 to 30, for example 24, ethylene glycol units (-O-CH2-CH2-).

[0142] According to one embodiment of the present invention, L comprises a linker selected from the group consisting of the following formulas 3 to 5: <Chemical formula 3>

[0143] TIFF2025508737000012.tif52170

[0144] <Chemical formula 4>

[0145] TIFF2025508737000013.tif61170

[0146] <Chemical formula 5>

[0147] TIFF2025508737000014.tif123170

[0148] In the above chemical formula, the wavy line

[0149] TIFF2025508737000015.tif6170

[0150] indicates the site to which Ab or D binds, and preferably, Ab binds to the side of the wavy line on the left, and D binds to the side of the wavy line on the right.

[0151] The D is preferably a drug moiety selected from the group consisting of V-ATPase inhibitors, apoptosis promoters, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule inhibitors, auristatins, dolastatins, maytansinoids, MetAP (methionine aminopeptidase), inhibitors of nuclear export of protein CRM1, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reaction in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binders and DHFR inhibitors.

[0152] The antibodies, antibody fragments (e.g., antigen-binding fragments) or functional equivalents of the present disclosure may be conjugated to a drug moiety that modifies a given biological response. The drug moiety should not be construed as being limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein, peptide or polypeptide possessing a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin, proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokines, apoptotic agents, antiangiogenic agents, or biological response modifiers such as lymphokines.

[0153] In some embodiments, an antibody, antibody fragment (eg, an antigen-binding fragment), or functional equivalent of the present disclosure is conjugated to a drug moiety, such as a cytotoxin, a drug (eg, an immunosuppressant), or a radiotoxin. Examples of cytotoxins include taxanes (see, e.g., International (PCT) Patent Application Nos. WO 01 / 38318 and PCT / US03 / 02675), DNA-alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin E, auristatin F, maytansinoids, and cytotoxic agents including reactive polyethylene glycol moieties (see, e.g., Sasse et al., J. Antibiot. (Tokyo), 53, 879-85 (2000); Suzawa et al., Bioorg. Med. Chem., 8, 2175-84 (2000); Ichimura et al., J. Antibiot. (Tokyo), 44, 1045-53 (1991); Francisco et al., Blood 2003). 15;102(4):1458-65], U.S. Patent Nos. 5,475,092, 6,340,701, 6,372,738, and 6,436,931, U.S. Patent Application Publication No. 2001 / 0036923A1, pending U.S. Patent Application Serial Nos. 10 / 024,290 and 10 / 116,053, and International (PCT) Patent Application No. WO01 / 49698), taxon, cytochalasin B, gramicidin D, ethidium bromide, emthyl esterase, methionine, methyltransferase ... Examples of suitable anti-inflammatory drugs include, but are not limited to, rifabutin, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP), cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine) (e.g., Seattle Genetics, Inc., Seattle, WA). Genetics, see US20090304721).

[0154] Other examples of cytotoxins that can be conjugated to the antibodies, antibody fragments (antigen-binding fragments), or functional equivalents of the present disclosure include duocarmycins, calicheamicins, maytansines, and auristatins, and derivatives thereof.

[0155] Methods for conjugating various types of cytotoxins, linkers, and therapeutic agents to antibodies are known in the art, see, e.g., Saito et al., (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail et al., (2003) Cancer Immunol. Immunother. 52:328-337; Payne, (2003) Cancer Cell 3:207-212; Allen, (2002) Nat. Rev. Cancer 2:750-763; Pastan and Kreitman, (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter and Springer, (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0156] The antibodies, antibody fragments (e.g., antigen-binding fragments) or functional equivalents of the present disclosure can also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals called radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine-131, indium-111, yttrium-90 and ruthenium-177. Methods for producing radioimmunoconjugates are established in the relevant art. Zevalin TM (IDEC Pharmaceuticals) and Bexxar TM Exemplary radioimmunoconjugates are commercially available, including (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies disclosed herein. In a particular aspect, a macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are generally known in the relevant art and are described in the literature in: Denardo et al., (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med. Biol. 26(8):943-50, each of which is incorporated by reference.

[0157] In some embodiments, D is a microtubule inhibitor (MTI) moiety.

[0158] The microtubule inhibitor may include, but is not limited to, a drug selected from the group consisting of SN-38 ((4S)-4,11-diethyl-4,9-dihydroxy-1,4-dihydro-3H,14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione), auristatin, dolastatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), monomethydolastatin 10 (MMAD), or a combination thereof.

[0159] SN-38 inhibits DNA topoisomerase I, preventing cell division. It has lower potency and fewer side effects than previously used drugs, and can be used to increase the DAR and therefore the efficacy against side effects of antibody-drug conjugates.

[0160] The antibody or antigen-binding fragment thereof preferably specifically binds to an epitope in domain 2 / 3 of human c-Kit represented by SEQ ID NO:11 (i.e., D113 to D309), more preferably specifically binds to an epitope in domain 2 of human c-Kit represented by SEQ ID NO:9 (i.e., R122 to R205) and / or domain 3 of human c-Kit represented by SEQ ID NO:10 (i.e., S240 to H263), more preferably specifically binds to one or more amino acid residues selected from the group consisting of R122, Y125, R181, K203, and R205 in SEQ ID NO:9 and / or one or more amino acid residues selected from the group consisting of S261 and H263 in SEQ ID NO:10.

[0161] In some embodiments, the antibody or antigen-binding fragment thereof cross-competes with a reference antibody comprising a heavy chain variable domain set forth in SEQ ID NO:7 and a light chain variable domain set forth in SEQ ID NO:8 for binding to an epitope in domain 2 / 3 of human c-Kit of SEQ ID NO:11 (i.e., D113-D309).

[0162] According to one embodiment of the present invention, the antibody-drug conjugate is selected from the group consisting of the following formulas 6 to 8: <Chemical formula 6>

[0163] TIFF2025508737000016.tif45170

[0164] <Chemical formula 7>

[0165] TIFF2025508737000017.tif65170

[0166] <Chemical formula 8>

[0167] TIFF2025508737000018.tif105170

[0168] In the above formula, a is an integer of 1 to 8, and b and c are each an integer of 1 to 4.

[0169] According to another aspect of the present invention, there is provided an antibody-drug conjugate selected from the group consisting of the following formulas 6 to 8: <Chemical formula 6>

[0170] TIFF2025508737000019.tif45170

[0171] <Chemical formula 7>

[0172] TIFF2025508737000020.tif65170

[0173] <Chemical formula 8>

[0174] TIFF2025508737000021.tif104170

[0175] In the above formula, Ab is an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 of SEQ ID NO:1, a heavy chain CDR2 of SEQ ID NO:2, a heavy chain CDR3 of SEQ ID NO:3, a light chain CDR1 of SEQ ID NO:4, a light chain CDR2 of SEQ ID NO:5 and a light chain CDR3 of SEQ ID NO:6; a is an integer from 1 to 8, and b and c are each an integer from 1 to 4.

[0176] In some embodiments, the antibody-drug conjugate has the structure of Formula 7, wherein b is 2.

[0177] In some embodiments, the antibody-drug conjugate has the structure of Formula 7, wherein b is 4.

[0178] III. Composition According to yet another aspect, the present invention provides a composition comprising the antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0179] In some embodiments, the composition is a pharmaceutical composition for preventing or treating cancer.

[0180] According to yet another aspect of the invention, there is provided a composition for use in therapy.

[0181] In some embodiments, the application is in the treatment of cancer.

[0182] According to yet another aspect of the present invention, there is provided a use for the manufacture of said pharmaceutical composition.

[0183] According to yet another aspect of the present invention, there is provided a method for preventing or treating cancer, comprising administering the composition to a subject in need thereof.

[0184] Disclosed herein is a method of treating a tumor (or cancer) in a subject, the method comprising administering to the subject the antibody-drug conjugate or a pharma- ceutically acceptable salt thereof. In certain embodiments, the antibody-drug conjugate specifically binds to c-Kit protein and reduces c-Kit activity.

[0185] In some embodiments, the methods disclosed herein inhibit and / or reduce tumor growth in a subject. In certain embodiments, tumor growth (e.g., tumor volume or weight) is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to a baseline (e.g., a corresponding tumor volume or weight in a subject that has not received a composition disclosed herein, e.g., the antibody drug conjugate). In some embodiments, the methods disclosed herein increase the mean tumor growth inhibition (TGI) by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to a baseline (e.g., a corresponding tumor volume or weight in a subject that has not received a composition disclosed herein, e.g., the antibody drug conjugate). In some embodiments, administration of a composition disclosed herein (e.g., the antibody drug conjugate) increases mean survival by at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or more, compared to a baseline (e.g., a corresponding value in a subject who did not receive a composition disclosed herein, e.g., the antibody drug conjugate).

[0186] In some embodiments, tumors that can be treated with the methods disclosed herein are derived from cancers that are typically responsive to existing anti-cancer drugs and cancers that are typically unresponsive to existing anti-cancer drugs. In some embodiments, the cancer is a solid tumor or a cancer with a hematological malignancy (liquid tumor).

[0187] Non-limiting examples of cancers requiring treatment include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, pancreatic cancer, cervical cancer, Gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (or carcinoma), germ cell tumors, childhood sarcoma, nasal natural killer, melanoma (e.g., metastatic malignant melanoma, e.g., cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, colon cancer, mast cell tumors, endocrine system cancer, parathyroid cancer, adrenal cancer, sarcoma of soft tissue, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis cancer, tumor angiogenesis, pituitary adenoma, Kaposi's sarcoma, epithelial cancer, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, virus-associated or virus-derived cancers (e.g., human papillomavirus (HPV-associated or derived tumors), as well as the two major blood cell lineages, i.e., Hematological malignancies derived from either myeloid cell lines (which produce granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lines (which produce B, T, NK, and plasma cells), e.g., all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphoid, and / or myeloid leukemias, e.g., acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma;Lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoblastic lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), lymphoid hematopoietic neoplasms , acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoblastic lymphoma with Waldenström's macroglobulinemia (LPL); myelomas, e.g., IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma, myeloma (also called inactive myeloma), solitary plasmocytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; myeloid hematopoietic neoplasms, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminomas, teratocarcinomas, tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma, lymphoid hematopoietic neoplasms, including T-cell and B-cell neoplasms, including T-cell disorders such as T-cell prolymphocytic leukemia (T-PLL), including, but not limited to, small cell and gyrus cell types; T-cell large granular lymphocytic leukemia (LGL); a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric T-cell lymphoma; head and neck, renal, rectal, and thyroid cancers;acute myeloid lymphoma, and any combination thereof;

[0188] According to one embodiment of the invention, the cancer is one or more cancers selected from the group consisting of esophageal cancer, gastric cancer, GIST, colon cancer, rectal cancer, lung cancer, particularly SCLC, breast cancer, mast cell tumor and leukemia, particularly AML.

[0189] In some embodiments, the methods disclosed herein may also be used to treat metastatic cancer, unresectable, refractory cancer (e.g., cancer that is resistant to existing cancer therapies, e.g., immunotherapy, e.g., therapy with blocking PD-(L)1 antibodies), and / or recurrent cancer. In certain embodiments, the methods disclosed herein may be used to treat recurrent cancer.

[0190] In some embodiments, the methods disclosed herein can be administered in combination with existing cancer therapies, including chemotherapy, such as imatinib and other c-KIT pathway inhibitors.

[0191] In some embodiments, the methods disclosed herein effectively increase the survival duration of a subject (e.g., tumor-affected), e.g., the survival duration of a subject is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year or more, when compared to a reference individual (e.g., another subject not treated with a composition disclosed herein, e.g., the antibody drug conjugate). In yet other embodiments, the methods disclosed herein increase the survival duration of a subject by a greater level (about 1 month greater, about 2 months greater, about 3 months greater, about 4 months greater, about 5 months greater, about 6 months greater, about 7 months greater, about 8 months greater, about 9 months greater, about 10 months greater, about 11 months greater, or about 1 year greater) than the survival duration of a reference subject (e.g., another subject not treated with a composition disclosed herein, e.g., the antibody drug conjugate).

[0192] In some embodiments, the methods of the invention increase the duration of disease progression-free survival of a subject, e.g., the duration of disease progression-free survival of a subject is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or up to at least about 1 year, as compared to a reference subject (e.g., another subject not treated with a composition disclosed herein, e.g., the antibody drug conjugate).

[0193] In some embodiments, the methods disclosed herein effectively increase the response rate in a group of subjects, e.g., the response rate in a group of subjects is increased by at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%, when compared to a reference subject (e.g., other subjects not treated with a composition disclosed herein, e.g., the antibody drug conjugate).

[0194] In some embodiments, the subject treated with the methods is a non-human animal, such as a rat or a mouse. In some embodiments, the subject treated with the methods is a human.

[0195] The present invention also includes methods of treating cancer in a subject in combination with other cancer agents. In some embodiments, the antibody drug conjugates useful in the methods of the present invention can be administered in combination therapy, i.e., in combination with at least one other anti-cancer agent and / or immunomodulatory agent, e.g., T cell stimulating (e.g., activating) agent. In some embodiments, the antibody drug conjugates useful in the methods of the present invention can be administered in combination with other compounds, drugs, and / or agents used to treat cancer. Such compounds, drugs, and / or agents may include, for example, chemotherapy drugs, small molecule drugs, or antibodies that stimulate an immune response against cancer. In some embodiments, the methods disclosed herein are used in combination with standard therapies (e.g., surgery, radiation, and chemotherapy). In other embodiments, the methods disclosed herein are used as maintenance therapies, e.g., therapies that attempt to prevent tumor development or recurrence.

[0196] In some embodiments, the antibody drug conjugates disclosed herein may be used in combination with one or more additional cancer agents, including immunotherapeutic agents, chemotherapeutic agents, targeted therapeutic agents, radiotherapeutic agents (radiotherapy), or any combination thereof.

[0197] Disclosed herein are compositions comprising the antibody-drug conjugates disclosed herein with the desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, zeaxanthin, erythritol, erythritol, arginine ... amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0198] In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate disclosed herein in a pharma- ceutical acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of the antibody-drug conjugate disclosed herein, and optionally one or more additional prophylactic or therapeutic agents in a pharma- ceutical acceptable carrier. In some embodiments, the antibody-drug conjugate is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical composition disclosed herein is useful for reducing c-Kit activity, which can be used to treat cancer.

[0199] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents for metal ions, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactate Ringer's injection. Non-aqueous vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antibacterial agents in bacteriostatic or fungistatic concentrations may be added to parenteral formulations packaged in multi-dose containers, and these include phenol or cresol, mercury-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonicity agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0200] The pharmaceutical composition may be formulated to suit any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, parenteral, intraspinal, intraventricular, pulmonary, subcutaneous, or intraventricular routes. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated. Injectables may be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other formulations, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0201] Formulations for parenteral administration of antibody drug conjugates include sterile ready-to-inject solutions, sterile dry soluble products ready for combination with a solvent immediately prior to use, including tablets for subcutaneous injection, e.g., lyophilized powders, sterile ready-to-inject suspensions, sterile dry insoluble products ready for combination with a vehicle immediately prior to use, and sterile emulsions. The solutions can be aqueous or non-aqueous.

[0202] The antibody-drug conjugate or its pharma-ceutically acceptable salt disclosed herein can also be formulated to target specific tissue, receptor, or other body area of ​​the subject to be treated.Various targeting methods are known to those skilled in the art.It is considered that such targeting methods are used in the present composition. Non-limiting examples of targeting methods are described in U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874. In certain embodiments, the antibody-drug conjugates or pharma- ceutically acceptable salts thereof disclosed herein can be used to treat cancer.

[0203] Compositions to be used for in vivo administration can be sterilized, for example by filtration through sterile filtration membranes.

[0204] According to yet another aspect of the present invention, there is provided a composition for diagnosing cancer, comprising the antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0205] IX. Kit A kit is disclosed herein that includes one or more antibody-drug conjugates or pharma-ceutically acceptable salts thereof disclosed herein. In some embodiments, a pharmaceutical pack or kit is disclosed herein that includes one or more containers filled with one or more of the components of the compositions disclosed herein that include one or more antibody-drug conjugates or pharma-ceutically acceptable salts thereof disclosed herein, and optional instructions for use. In some embodiments, the kit contains the pharmaceutical composition disclosed herein and any of the prophylactic or therapeutic agents disclosed herein. In some embodiments, the kit is disclosed as a cancer diagnostic composition that includes the composition disclosed herein and instructions for detection and / or diagnosis methods.

[0206] In some embodiments, a method of providing information for the diagnosis of cancer is provided that includes treating a sample isolated from a subject with an antibody-drug conjugate or a pharma- ceutically acceptable salt thereof.

[0207] X. Production Method Disclosed herein are methods of producing one or more of the anti-c-Kit antibody drug conjugates disclosed herein.

[0208] The conjugates of the present disclosure can be prepared by any method known in the relevant art, such as those described in U.S. Patent Nos. 7,811,572, 6,411,163, 7,368,565, and 8,163,888, and U.S. Patent Publication Nos. 2011 / 0003969, 2011 / 0166319, 2012 / 0253021, and 2012 / 0259100, the entire teachings of which are incorporated herein by reference.

[0209] In addition, methods for conjugating various types of cytotoxins, linkers, and drugs to antibodies are known in the relevant art, see, for example, the literature (Saito et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail et al., Cancer Immunol. Immunother. 52:328-337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091 (2002); Senter and Springer, Adv. Drug Deliv. Rev. 53:247-264 (2001)).

[0210] In some embodiments, the method includes producing an antibody or antigen-binding fragment (Ab) that specifically binds to human c-Kit (L1). m -(S)n -(L2) p -(D) q 4. The method of claim 1, further comprising the step of: In the above formula, L1 is a linker connecting the Ab and S or the Ab and L2; S is a spacer with or without a polymer attached; L2 is a cleavable linker; D is a drug moiety; m is an integer from 0 to 8; n is an integer from 0 to 8; p is an integer from 1 to 8; q is an integer from 1 to 8.

[0211] The definitions and related explanations of the above terms are the same as those described in I. Definitions and II. Antibody Drug Conjugates (ADCs).

[0212] In some embodiments, the method comprises conjugating a linker (L)-drug (D) to an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6, wherein L comprises a cleavable linker.

[0213] In some embodiments, D is a microtubule inhibitor moiety.

[0214] In some specific examples, the m is 1-4, the n is 1-4, the p is 1-4, and the q is 1-8. Effect of the Invention

[0215] The features and advantages of the present invention can be summarized as follows: (i) The present invention provides an antibody-drug conjugate comprising an antibody that specifically binds to human c-Kit, a cleavable linker, and a microtubule inhibitor moiety.

[0216] (ii) The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises the antibody-drug conjugate as an active ingredient.

[0217] (iii) The antibody-drug conjugate of the present invention has a strong anticancer activity against c-Kit positive and negative cancer cell lines and / or imatinib-resistant cancer cell lines, and is characterized by its ability to induce complete remission. [Brief description of the drawings]

[0218] Figure 1 shows a method for preparing an ADC using a cleavable linker. Figure 1a shows the linker-drug used in one example, and Figure 1b shows the schematic process for ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u).

[0219] FIG. 2 shows the results of an in vitro cell viability assay to confirm the efficacy of the ADCs of the present invention in c-Kit positive and negative cancer cell lines.

[0220] FIG. 3 shows the results of confirming the in vivo efficacy of the ADC of the present invention against GIST in mice (GIST T1 cells) (FIG. 3a shows the results for PoC-DM1; FIG. 3b shows the results for 003-1, 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 2; FIG. 3c shows the results for 004-2, 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 4; and FIG. 3d shows the results for 003-2, 2G4-MC-Val-Cit-PAB-MMAE).

[0221] FIG. 4 shows the results of confirming the in vivo efficacy of the ADC of the present invention against GIST in mice (GIST-430 / 654 cells) (FIG. 4a shows the results for PoC-DM1; FIG. 4b shows the results for 003-1, 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 2; FIG. 4c shows the results for 004-2, 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 4; FIG. 4d shows the results for 003-2, 2G4-MC-Val-Cit-PAB-MMAE; and FIG. 4e shows the results for 007-1, 2G4-ThioBridge®-Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2).

[0222] FIG. 5 shows the results of confirming the in vivo efficacy of the ADC of the present invention against SCLC in mice (SCLC-H526 cells) (FIG. 5a shows the results for PoC-DM1; FIG. 5b shows the results for 003-1, 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 2; FIG. 5c shows the results for 004-2, 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 4; and FIG. 5d shows the results for 003-2, 2G4-MC-Val-Cit-PAB-MMAE).

[0223] FIG. 6 shows the results of confirming the in vivo efficacy of the ADC of the present invention against mast cell tumors in mice (HMC 1.2 cells).

[0224] FIG. 7 shows the results confirming the efficacy of the ADC of the present invention upon tumor reinoculation.

[0225] FIG. 8 shows the results of confirming the in vivo efficacy of the ADC of the present invention against AML in mice (Kasumi-1 cells).

[0226] FIG. 9 shows the results of confirming the in vivo efficacy of the ADC of the present invention against breast cancer in mice (MDA-MB-468 cells) (FIG. 9a shows the results for PoC-DM1; FIG. 9b shows the results for 003-1, 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 2; FIG. 9c shows the results for 004-2, 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 4; and FIG. 9d shows the results for 003-2, 2G4-MC-Val-Cit-PAB-MMAE).

[0227] FIG. 10 shows the results of a re-challenge assay against SCLC in mice (SCLC-H526 cells) against the ADCs of the present invention.

[0228] FIG. 11 shows the results of a re-challenge assay of mouse mastocytoma (HMC 1.2 cells) against the ADCs of the present invention.

[0229] FIG. 12 shows the results of a preliminary in vivo toxicity analysis of the ADC of the present invention when administered at a high concentration (10 mg / kg).

[0230] FIG. 13 shows the results of in vivo preliminary toxicity analysis of the ADC of the present invention at higher doses (20 / 40 / 60 mg / kg) (FIG. 13a shows the results for the 2G4 antibody; FIG. 13b shows the results for PoC-DM1; FIG. 13c shows the results for 003-1, 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 2; FIG. 13d shows the results for 004-2, 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 4).

[0231] Figure 14 shows the docking model of 2G4 Fab:c-Kit. (Figure 14a) c-Kit mutants are summarized. (Figure 14b and Figure 14c) c-Kit wild type and mutant proteins were purified and serially diluted and then coated on 96-well plates. The binding of 2G4 was investigated by ELISA. (Figure 14d) The structural model of the 2G4 Fab:c-Kit complex with the highest score calculated by HADDOCK is shown. (Figure 14f) The heavy (H) variable (V) and constant (C) regions and the light (L) variable (V) and constant (C) regions of 2G4 are represented by VH, CH1, VL and CL, respectively. (Figure 14e) Stick representation of the interface between 2G4 and c-Kit is shown. Residues of the D2 and D3 regions of c-Kit are represented by shaded bars, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0232] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0233] Working Example Manufacturing Example Production Example 1. Production of anti-c-Kit antibody 2G4 Production Example 1-1. Preparation of immunized mice 50μg (per mouse) of recombinant c-Kit protein (cat# PKSH030939) purchased from Elabscience was mixed with the same volume of Freund's complete adjuvant (Sigma, USA) to prepare an emulsion. The emulsion thus prepared was injected intraperitoneally into six 7-week-old female humanized NSG mice produced by injection of human CD34+ cells. 50μg of antigen was injected into each mouse in a total volume of 500μl. After 1 week and 2 weeks, emulsions mixed with antigen and incomplete Freund's adjuvant (Sigma, USA) were further injected intraperitoneally into the mice.

[0234] Example 1-2. Confirmation of antibody production Blood was collected from the eyeballs of the immunized mice through the above method, placed in a 1.5 mL microcentrifuge tube, and centrifuged at 13,000 rpm for 10 minutes. Serum was separated and stored at -20°C until an experiment to confirm antibody production was performed. After confirming antibody production by performing an enzyme immunoassay using the antigen protein, an emulsion of incomplete Freund's adjuvant (Sigma, USA) and the antigen was further injected intraperitoneally into the mice 3 days before cell fusion.

[0235] Production Example 1-3. Production of hybridomas After confirming antibody production, the mice were sacrificed, and spleen cells were isolated and fused with myeloma cells P3X63Ag 8.653 (ATCC CRL-1580) to produce hybridomas.

[0236] Specifically, mouse P3X63Ag 8.653 cells were cultured in a culture plate using RPMI 1640 medium supplemented with 10% fetal bovine serum. To perform cell fusion, P3X63Ag 8.653 cells were washed twice with serum-free RPMI 1640 medium (Hyclone, USA) and cultured at 1x10 7 The cell concentration was adjusted to 100%. Mice were sacrificed by cervical dislocation, and the spleens were harvested and then placed in a mesh container (Sigma, USA) to separate the cells. After preparing a suspension of spleen cells, the suspension was washed using centrifugation. The spleen cell solution was exposed to Tris-NH4Cl (TRIS 20.6g / L, NH4Cl 8.3g / L) to lyse red blood cells. The completely separated antibody-producing cells were centrifuged at 400xg for 5 minutes. They were then washed twice with serum-free medium and resuspended in 10mL medium. Lymphocytes were counted using a hemocytometer, and lymphocytes were 1x10 8 were mixed with 1x10 P3X63Ag 8.653 cells (10:1) in serum-free medium.

[0237] After centrifugation at 400xg for 5 min, 1mL of the solution was added dropwise using 50% (M / V) polyethylene glycol 1500 (Sigma, USA) warmed at 37°C and mixed for 1 min. The fusion mixture thus prepared was diluted with serum-free RPMI1640 and centrifuged at 400xg for 3 min. The cells were suspended in 35mL of RPMI1640 selection medium supplemented with 20% fetal bovine serum and HAT (100μM hypoxanthine, 0.4μM aminopterin, 16μM thymidine). 100μl of the suspension was loaded onto a 96-well plate coated with feeder cells (macrophages isolated from the peritoneal cavity using RPMI1640) 1 day before and cultured at 37°C, 5% CO2. After 5 days, the HAT medium was replaced at intervals of 2-3 days, and the cells were cultured for 14 days. After 14 days, the medium was replaced with RPMI1640 supplemented with 20% fetal bovine serum and HT (medium obtained by removing 0.4 μM aminopterin from HAT), and secondary culture was performed.

[0238] Production Example 1-4. Selection and separation of antibody-producing fused cells The supernatant of the fused cell culture prepared above was collected, and enzyme immunoassay was performed to confirm the production of specific antibodies against the prepared antigen. The culture medium of the fused cells showing an appropriate concentration of 4 times or more compared to the negative control group was selected, transferred to a 24-well plate and cultured. Furthermore, after diluting the cells to a limiting dilution so that one cell was placed per well in a 96-well plate and culturing, the culture medium was collected, and the c-KIT protein used as the antigen was coated on the 96-well plate at 0.1 μg per well. Then, enzyme immunoassay was performed to select fused cells that finally produced 15 monoclonal antibodies (1C6, 1H2, 1A6, AFA, 2B3, 2G4, 4G5, 4C4, 4C7, 4D7, 1E1, 2H6, 1G3, 1A3, 1D3).

[0239] Example 1-5. Selection of c-KIT antibody To confirm the c-KIT binding ability of the selected antibodies, SPR (Surface Plasmon Resonance) was performed. 20 μg of human c-Kit (elabscience, PKSH030939), 20 μg of mouse c-Kit (SB, Lot#LC05DE2304), and 20 μg of rat c-Kit (SB, Lot#LC06SE1787), which were used for antibody production, were immobilized on a PEG (Reichert, USA) chip using SR7500DC (Reichert, USA). Then, the antibodies were run at different concentrations, and the KD value of the affinity for c-Kit was analyzed using the Scrubber2 program. The KD value is the value obtained by dividing kd by ka, and the lower the value, the greater the binding ability to the target.

[0240] As a result, the KD value of the 2G4 antibody for human c-Kit was approximately 2.8237 (±0.9)X10 -12 It showed a strong affinity for M. It showed the highest affinity for humans, followed by mice and rats.

[0241] The CDR sequences of the selected 2G4 antibodies are shown in Table 1 below:

[0242] [Table 1]

[0243] The sequences of the heavy and light chain variable regions of the 2G4 antibody are as shown in Table 2 below:

[0244] [Table 2]

[0245] Manufacturing example 2. Manufacturing of “2G4-SMCC-DM1” (#POC-DM1) 2G4-ADC (antibody-drug conjugate) was prepared containing SMCC (N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate) as a non-cleavable linker and DM1 (N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine) as a microtubule inhibitor. The conjugates of linker-payload to 2G4 were measured using a UV-Vis spectrophotometer, and the drug-antibody ratio (DAR) was determined through LC-MS analysis.

[0246] Specifically, 2G4 and human IgG1 (Sino Biological) were dialyzed against conjugation buffer (0.1 M sodium phosphate and 0.15 M NaCl, pH 7.2) and conjugated with SMCC-DM1 (MedChemExpress) at 1:5 or 1:10 molar ratio for 1.5 h at RT. Fractionation was performed to remove unbound SMCC-DM1 and aggregated 2G4-DM1. The antibody fractions were pooled and dialyzed against formulation buffer (10 mM sodium succinate, 0.05% polysorbate 20, and 6% sucrose; pH 5.0). The optical density (OD) at 280 nm and 252 nm was examined using a SPECTROstar Nano (BMG LABTECH) to determine the presence or absence of conjugation. The DAR was determined using liquid chromatography-tandem mass spectrometry (LC-MS) analysis.

[0247] To confirm the conjugation of DM1 and 2G4, the absorbance of naked 2G4 and ADC was analyzed using a UV-Vis spectrophotometer at 252 nm, because DM1 absorbs ultraviolet light at 252 nm, and the absorbance of naked 2G4 and 2G4-DM1 was compared. The absorbance of 2G4-DM1 was higher than that of 2G4 at 252 nm. The results indicated that DM1 was potentially conjugated to 2G4.

[0248] Mass spectrometry analysis of 2G4-DM1 confirmed that it was composed of a diverse population with a range of 1 to 4 drugs per antibody, and the average DAR of 2G4-DM1 was 1.8.

[0249] Preparation Example 3. Preparation of ADCs using cleavable linkers Four ADCs were produced using cleavable linkers. Specifically, ADCs were produced with classical maleimide conjugation or ThioBridge® (disulfide rebridging conjugation technology, Abzena, USA) to link MMAE and SN-38, with average DARs ranging between 2 and 8. A proportion of ThioBridge® MMAE ADCs with average DARs of 2 and 4 were produced along with a maleimide MMAE control ADC with an average DAR of 4. The average DAR of ThioBridge® SN-38 ADC was 8. A cathepsin-cleavable "Val-Cit-PAB" motif was included in all linkers to allow lysosomal release of the payload (Figure 1a). In the case of ThioBridge® SN-38 ADC, the carbonate bond connecting SN-38 to the linker is sensitive to hydrolysis, allowing for a pH-based payload release mechanism. ADCs with high monomer purity (>97%) were prepared in 29-56 mg quantities for subsequent biological testing. The preparation process for ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) is shown in Figure 1b.

[0250] Preparation Example 3-1. Preparation of 2G4-MC-VC-PAB-MMAE mAb 2G4 at 13.38 mg / mL in Dulbecco's PBS, pH 7.4, 5 mM EDTA (5.232 mL; 70.0 mg; 470 nmol; 1.0 equiv.) was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (8.505 mL). A 5 mM solution of TCEP in endotoxin-free water (263.4 μL, 1317 nmol, 2.8 equiv.) was added to the mAb 2G4 dilution. Reduction proceeded for 2 h at 40° C. for a final antibody concentration of 5.0 mg / mL.

[0251] After 2 h at 40 °C, the reduction mixture was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (2.450 mL) and further diluted in DMF (177 μL) after cooling to 22 °C. A 4.26 mg / mL (3.23 mM) solution of MC-VC-PAB-MMAE in DMF was prepared by the addition of 3.95 mg (3.00 μmol) of MC-VC-PAB-MMAE (MW = 1317 g.mol -1 ) in 927 μL of DMF. The MC-VC-PAB-MMAE solution in DMF (873 μL; 3.72 mg; 2823 nmol; 6.0 equiv.) was added to the reduced mAb 2G4 solution for a final concentration of 6% DMF and a final antibody concentration of 4.0 mg / mL. The conjugation reaction was allowed to proceed for 1 h at 22° C.

[0252] After 1 h at 22 °C, the reaction mixture was buffer exchanged by spin filtration using a Zebaspin column (7 kDa MWCO; 10 mL) equilibrated with 20 mM histidine, 50 mM NaCl, 5% sucrose, pH 6.5 according to the manufacturer's guidelines. The recovered conjugate sample was further polished and concentrated to 7.13 mg / mL by UF / Diafiltration using a Vivaspin 20 centrifugal concentrator (PES membrane, 30 kDa MWCO) equilibrated with 20 mM histidine, 50 mM NaCl, 5% sucrose, pH 6.5. The concentrated conjugate sample (56.3 mg; 7.90 mL) was sterile filtered through a 0.22 μm pore size PVDF membrane filter.

[0253] The antibody-drug conjugate obtained through this was named "2G4-MC-VC-PAB-MMAE" and the conjugate was characterized by HIC and SEC. 56 mg of 2G4-MC-VC-PAB-MMAE ADC was isolated with high monomer content (>98%, SEC) and an average DAR of 4.2 (HIC) (hereinafter referred to as 003-2).

[0254] Preparation Example 3-2. Preparation of 2G4-ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 2 mAb 2G4 at 14.77 mg / mL in Dulbecco's PBS, pH 7.4, 5 mM EDTA (2.180 mL; 32.2 mg; 216 nmol; 1.0 equiv.) was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (4.148 mL). A 5 mM solution of TCEP in endotoxin-free water (112 μL, 562 nmol, 2.6 equiv.) was added to the diluted mAb 2G4 solution. Reduction proceeded for 2 h at 40° C. for a final antibody concentration of 5.0 mg / mL.

[0255] After 2 hours at 40° C., the reduction mixture was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (805 μL), cooled to 22° C., and further diluted with DMF (403 μL). A 4.52 mg / mL (1.61 mM) solution of ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) in DMF was prepared by the synthesis of 2.15 mg (768 nmol) of ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (MW=2805.0 g.mol -1 ) in 477 μL of DMF. A solution of ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (402 μL; 1.82 mg; 648 nmol; 3.0 eq.) in DMF was added to the reduced 2G4 solution to give a final DMF concentration of 10% and a final antibody concentration of 4.0 mg / mL. The conjugation reaction proceeded for 18 hours at 22° C.

[0256] After 18 h at 22 °C, the reaction mixture was buffer exchanged by spin filtration using a Zebaspin column (7 kDa MWCO; 10 mL) equilibrated with 20 mM histidine, 50 mM NaCl, 5% sucrose, pH 6.5 according to the manufacturer's guidelines. The recovered conjugate sample was further polished and concentrated to 7.25 mg / mL by UF / DF using a Vivaspin 20 centrifugal concentrator (PES membrane, 30 kDa MWCO) equilibrated with 20 mM histidine, 50 mM NaCl, 5% sucrose, pH 6.5. The concentrated conjugate sample (29.7 mg; 4.10 mL) was sterile filtered through a 0.22 μm pore size PVDF membrane filter.

[0257] The antibody-drug conjugate obtained through this was named "2G4-ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 2" and the conjugate was characterized by HIC and SEC. 30 mg of 2G4-ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR2 ADC was isolated with high monomer content (>97%, SEC) and an average DAR of 2.0 (HIC) (hereinafter referred to as 003-1).

[0258] Preparation Example 3-3. Preparation of 2G4-ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 4 mAb 2G4 at 20.71 mg / mL in Dulbecco's PBS, pH 7.4, 5 mM EDTA (5.794 mL, 120 mg, 806 nmol, 1.0 equiv.) was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (17 mL). A 5 mM solution of TCEP in endotoxin-free water (967.2 μL, 4836 nmol, 6.0 equiv.) was added to the diluted mAb 2G4 solution. Reduction proceeded at 40° C. for 1 h at a final antibody concentration of 5.0 mg / mL.

[0259] After 1 h at 40° C., the reduction mixture was diluted with propylene glycol (6.40 mL) and cooled to 22° C. An 8.48 mg / mL (3.02 mM) solution of ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) in DMF was obtained by eluting ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (MW=2805.0 g.mol -1 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) solution (1600 μL; 13.57 mg; 4836 nmol; 6.0 equiv.) in DMF was added to the reduced 2G4 solution, yielding 5% DMF, 20% propylene glycol and a final antibody concentration of 3.8 mg / mL. The conjugation reaction proceeded for 18 hours at 22°C.

[0260] After 18 h at 22 °C, the reaction mixture was diluted with 4 M sodium chloride, 50 mM sodium phosphate pH 7.0 (32.0 mL). The diluted reaction mixture was loaded onto a Proteus FliQ 10 mL column packed with HIC ToyoPearl® Phenyl-650S resin equilibrated with Buffer A (2.0 M sodium chloride, 50 mM sodium phosphate, pH 7.0 buffer). Elution was performed with Buffer B (50 mM sodium phosphate, 20% isopropanol, pH 7.0) at a constant flow rate of 1.5 mL / min and a 200 mL gradient from 0 to 100%. Fractions were analyzed by HIC and SEC and pooled based on DAR 4 species content (>90%). The pooled fractions were buffer exchanged and concentrated to 7.93 mg / mL by UF / DF using Vivaspin 20 centrifugal concentrators (PES membrane, 30 kDa MWCO) equilibrated with 20 mM histidine, 50 mM NaCl, 5% sucrose, pH 6.5. The concentrated conjugate sample (47.4 mg; 5.98 mL) was sterile filtered through a 0.22 μm pore size PVDF membrane filter.

[0261] The antibody-drug conjugate obtained through this was named "2G4-ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR 4 conjugate" and the conjugate was characterized by HIC and SEC. 47 mg of 2G4-ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u)DAR4 ADC was isolated with high monomer content (>98%, SEC) and an average DAR of 4.0 (HIC) (hereinafter referred to as 004-2).

[0262] Preparation Example 3-4. Preparation of 2G4-ThioBridge®-Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 mAb 2G4 at 18.71 mg / mL in Dulbecco's PBS, pH 7.4, 5 mM EDTA (3.474 mL, 65.0 mg, 437 nmol, 1.0 equiv.) was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (9.00 mL). A 5 mM solution of TCEP in endotoxin-free water (524.0 μL; 2620 nmol, 6.0 equiv.) was added to the diluted mAb 2G4 solution. Reduction proceeded for 1 h at 40 °C for a final antibody concentration of 5.0 mg / mL.

[0263] After 1 h at 40° C., the reduction mixture was diluted with propylene glycol (3.71 mL), cooled to 22° C., and further diluted with DMF (655 μL). A 10.0 mg / mL (2.18 mM) solution of ThioBridge®-Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 in DMF was prepared by dissolving 14.09 mg (3072 nmol) of ThioBridge®-Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 (MW=4588.0 g.mol−1) in 1409 μL of DMF. ThioBridge®-Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 was added to the reduced 2G4 solution in DMF (1202 μL; 12.02 mg; 2620 nmol; 6.0 equiv.) to yield 10% DMF, 20% propylene glycol and a final antibody concentration of 3.5 mg / mL. The conjugation reaction proceeded for 18 h at 22° C.

[0264] After 18 h at 22° C., the reaction mixture was diluted with 4 M sodium chloride, 50 mM sodium phosphate pH 7.0 (18.6 mL). The diluted reaction mixture was loaded onto a Proteus FliQ 10 mL column packed with HIC ToyoPearl® Phenyl-650S resin equilibrated with Buffer A (2.0 M sodium chloride, 50 mM sodium phosphate, pH 7.0 buffer). Elution was performed with Buffer B (50 mM sodium phosphate, 20% isopropanol, pH 7.0) at a constant flow rate of 1.5 mL / min and a 200 mL gradient of 0-100%. Fractions were analyzed by HIC and SEC and pooled by DAR 4 content (>80%). The pooled fractions were buffer exchanged and concentrated to 4.01 mg / mL by UF / DF using Vivaspin 20 centrifugal concentrators (PES membrane, 30 kDa MWCO) equilibrated with 20 mM histidine, 50 mM NaCl, 5% sucrose, pH 6.5. The concentrated conjugate sample (29.0 mg; 7.22 mL) was sterile filtered through a 0.22 μm pore size PVDF membrane filter.

[0265] The antibody-drug conjugate obtained through this was named "2G4-ThioBridge®-Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 conjugate" and the conjugate was characterized by LC-MS and SEC. 29 mg of 2G4-ThioBridge®-Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 was isolated with a high monomer content (>98%, SEC) and an average DAR of 7.4 (LC-MS) (hereinafter referred to as 007-1).

[0266] Analysis method (1)LC-MS analysis LC-MS analysis was performed using a POROSHELL 300SB C3 column (2.1x12.5mm, 5μm) coupled to a Waters XEVO G2S TOF mass spectrometer and a Waters Acquity H Class UPLC system. The mobile phase was buffer A (water, 0.1% formic acid). A gradient (2.5 min, 10% B, 10-80% B, gradient 3.5 min) was applied using buffer B (acetonitrile, 0.1% formic acid) at a flow rate of 0.4mL / min. The column was maintained at 60°C during the analysis. Maleimide ADCs were analyzed after reduction (10mM DTT, 40°C for 1 h). All ADCs were analyzed after dilution to 0.2mg / mL. 10μL of ADC solution was injected for analysis. The average DAR was calculated as a weighted average of the observed DAR species based on the major glycoform signal intensity (SI) of the deconvoluted m / z spectrum for non-reduced samples and the signal intensities for the light chain (LSI) and heavy chain (HSI) for reduced samples:

[0267] TIFF2025508737000024.tif30170

[0268] (2)SEC analysis Analytical SEC was performed using an ACQUITY UPLC BEH SEC column (4.6mmx15cm, 200Å, 1.7μm) and guard column (4.6mmx3cm) coupled to a Dionex Ultimate 3000 UPLC system. The mobile phase was 0.2M potassium phosphate buffer, pH 6.8, 0.2M potassium chloride, 15% (v / v) isopropanol. The flow rate was kept constant at 0.35mL / min. The column was maintained at 30°C during the analysis. The analysis was performed with isocratic elution for 10 min with UV detection at 248nm, 280nm and 365nm. 10μg of ADC was injected for analysis. The percentage of high molecular weight (HMW) species was calculated by comparing the peak area corresponding to HWM species at 280nm to the total peak area corresponding to all HWM and monomeric species at 280nm. The presence of free reagent-related species was assessed by comparing chromatographic traces of ADC and buffer samples in the low molecular weight species region of the chromatogram for retention times greater than 6 minutes at wavelengths corresponding to the maximum absorption of the payload (λ = 248 nm for MMAE, λ = 365 nm for SN-38).

[0269] (3)HIC analysis Analytical HIC was performed using a TOSOH Bioscience TSKgel Butyl-NPR column (4.6 mMx3.5 cm, 2.5 μm) coupled to a Dionex Ultimate 3000 UPLC system. The mobile phase was Buffer A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0. A linear gradient (0-100% B in 10.5 min) was applied to elute the bound species using Buffer B (20% isopropanol, 50 mM sodium phosphate, pH 7.0) at a flow rate of 1.35 mL / min. The column was maintained at 37 °C during the analysis. The analysis was performed with UV detection at 280 nm. 10 μg of ADC was injected per analysis. The percentage (i) of each DAR species was calculated by comparing the peak area of ​​each assigned peak to the total peak area. The average DAR was calculated as a weighted average of the observed DAR species based on the peak area under the curve (AUCi), and the average molecular weight of the ADC could be determined based on the DAR and the linker-payload mass contribution as follows:

[0270] TIFF2025508737000025.tif22170

[0271] Working Example Example 1. In vitro cell viability analysis To confirm the efficacy of the ADCs shown in Table 3 in c-Kit positive and negative cancer cell lines, an in vitro cell viability assay was performed.

[0272] Cells were seeded in 96-well cell culture black plates (Greiner Bio-One, Kremsmuenster, Austria) and cell numbers were scanned with a Celigo Imaging Cytometer instrument. Nine concentration points of ADC were prepared with 5- or 10-fold serial dilutions at starting concentrations of 40 or 200 μg / ml. After 3–5 days of treatment, cells were stained with Calcein-AM (1 μg / ml) or Hoechst 33342 (8 μM) fluorescent dye. Wells were scanned and analyzed using a Celigo Imaging Cytometer to count live cells. Cell viability was displayed graphically with GrapHPad Prism Software and half-maximal inhibitory concentration (IC50) values ​​were calculated (Table 4).

[0273] [Table 3]

[0274] [Table 4]

[0275] As can be seen from FIG. 2, the ADC group showed statistically significant efficacy compared to the control group.

[0276] Example 2. In vivo efficacy against GIST in mice (GIST T1 cells) Experiments were conducted to evaluate the efficacy of ADC agents against GIST. A GIST-T1 cell (c-Kit mutant and imatinib sensitive; Accession No.: CVCL_4976; c-Kit mutation site-Exon11 V560-L576) xenograft model was used.

[0277] Four-week-old female CB-17 SCID immunodeficient mice (Charles River Laboratories Japan, Inc.) were used in the experiment after being adapted to SPF (specific pathogen free) conditions for 12 days. GIST-T1 cells (5x106 The total injection volume containing the suspended cells was 200 μL.

[0278] On the 21st day after implantation, the average tumor volume was approximately 190 mm 3 Mice were enrolled on Day 0. After random assignment to one of six groups (n=5 / group), mice were administered ADC vehicle (5 mL / kg, ivtail), each ADC agent (0.5, 1.5 or 3.0 mg / kg, ivtail), imatinib (100 mg / kg, po), and ADC agent (3.0 mg / kg, ivtail) plus imatinib (100 mg / kg, po) (Table 5). The buffer for PoC-DM1 as the ADC vehicle was a mixture of 10 mM sodium succinate, 6% sucrose and 0.05% Tween 20 (pH 5.0), and for the rest the buffer was a mixture of 20 mM histidine (Merck, 104352), 50 mM sodium chloride (Sigma, S3014), 5% sucrose (Sigma, S0389) (PH 6.5), and 0.22 μM filter (Merck S2GPU11RE). Individual doses were calculated based on animal weight recorded immediately prior to dosing with a dose volume of 5 mL / kg body weight.

[0279] [Table 5]

[0280] The ADC agents administered are as follows in Table 6:

[0281] [Table 6]

[0282] All animals were observed daily for mortality, general condition and clinical signs (time, onset, severity and recovery). Tumor volume and body weight were measured twice weekly. To measure tumor volume, calipers were used to measure length, width and depth.

[0283] The results are shown in Figures 3a to 3d.

[0284] No abnormal symptoms were observed in any of the groups during the study before sacrifice, except for the imatinib 100 mg / kg group, in which one case of death occurred on Day 91.

[0285] Compared with the control group (vehicle), the imatinib 100 mg / kg group and the test ADC study groups (0.5 mg / kg, 1.5 mg / kg, 3.0 mg / kg, 3.0 mg / kg + imatinib 100 mg / kg) did not show a weight loss or weight gain of 10% or more for 56 days. After that, no rapid weight loss or rapid weight gain was observed in any of the groups. This implies that all of our ADC substances are very safe.

[0286] On day 56, compared to the control group (vehicle), the imatinib 100 mg / kg group suppressed tumor growth, while all ADC study groups at 3.0 mg / kg showed stronger tumor growth suppression effects. In particular, the 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (#003-1 and #004-2) ADC study groups showed significant tumor growth suppression effects even at 1.5 mg / kg.

[0287] Imatinib alone suppressed tumors but caused tumor recurrence after treatment was discontinued.Surprisingly, co-treatment of each ADC at 3 mg / kg with imatinib induced complete tumor remission without regrowth for up to 105 or 112 days after imatinib administration was discontinued.

[0288] After sacrifice, tumor weight was measured and, compared to the control group (vehicle), the imatinib 100 mg / kg group showed a reduction of about 59%, while all ADC study groups administered 1.5 mg / kg or more (excluding the #PoC-DM1 1.5 mg / kg group) showed a higher reduction potential (Table 7). In particular, the ADC study groups 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (#003-1 and #004-2) showed significant tumor growth suppression efficacy even in the 1.5 mg / kg group. When each ADC was administered at 3 mg / kg in combination with imatinib, most tumors disappeared.

[0289] [Table 7]

[0290] The above results confirm that the ADC group demonstrated statistically significant efficacy compared to the control group.

[0291] Example 3: In vivo efficacy against GIST in mice (GIST-430 / 654 cells) We also performed experiments to evaluate the efficacy of the ADC agent specifically against GIST resistant to imatinib, using a GIST-430 / 654 cell (imatinib-resistant, Exon 11 V560-L576+Exon 13 V654A) xenograft model.

[0292] Five-week-old female NOG (NOD / Shi-scid, IL-2RγKO) immunodeficient mice (CIEA Japan, Inc.) were acclimated for 12 days under specific pathogen-free (SPF) conditions before use in the experiment. Six-week-old mice were subcutaneously inoculated with GIST-430 / 654 cells (5Х10 6 / 100μL / Head / Serum free, IMDM medium) was subcutaneously injected. The total injection volume containing the suspended cells was 200μL.

[0293] Mice were enrolled in the study 21 days after implantation, with a mean tumor volume of approximately 180 mm 3 (Day 0). After random allocation to one of five groups (n=5 / group), mice were administered ADC vehicle (5 mL / kg, ivtail), each ADC substance (1, 3 or 5 mg / kg, ivtail) and imatinib (100 mg / kg, po) (Table 8). The buffer for PoC-DM1 as the ADC vehicle was a mixture of 10 mM sodium succinate, 6% sucrose and 0.05% Tween 20 (pH 5.0), and the buffer for the rest was a mixture of 20 mM histidine (Merck, 104352), 50 mM NaCl (Sigma, S3014), 5% sucrose (Sigma, S0389) (pH 6.5) and 0.22 μM filter (Merck, S2GPU11RE). Individual doses were calculated based on the animal's body weight recorded immediately prior to dosing with a dose volume of 5 mL / kg body weight.

[0294] [Table 8]

[0295] The ADC materials are as follows in Table 9:

[0296] [Table 9]

[0297] The sacrifice dates for each group are as follows: Vehicle and Imatinib 100 mg / kg: sacrifice on day 49.

[0298] -All ADC groups (except 007-1): Sacrifice on day 63.

[0299] - #007-1 group: sacrificed on day 42 (the #007-1 group was terminated before the control group (vehicle) so no comparison is made).

[0300] All animals were observed once daily for mortality, general condition and clinical signs (time, onset, severity and recovery). Tumor volume and body weight were measured twice weekly. To measure tumor volume, calipers were used to measure length, width and depth.

[0301] The results are shown in Figures 4a to 4e.

[0302] No abnormal symptoms were observed in any group during the study period prior to sacrifice.

[0303] No weight loss or weight gain of 10% or more was observed for 49 days between the imatinib 100 mg / kg group and the test ADC study groups (1 mg / kg, 3 mg / kg and 5 mg / kg) compared to the control group (vehicle). Thereafter, no rapid weight loss or rapid weight gain was observed in any of the groups. This suggests that all of the ADC materials of the present invention are very safe.

[0304] On day 49, compared to the control group (vehicle), the imatinib 100 mg / kg group showed little tumor growth inhibition, whereas all ADC study groups showed stronger tumor growth inhibition effects (except #007-1 1 mg / kg). In particular, the ADC study groups of 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (#003-1 and #004-2) were confirmed to have significant tumor growth inhibition effects in the 3 or 5 mg / kg groups, and tumors were maintained at almost the same level even after treatment was discontinued.

[0305] Tumor weights were measured after sacrifice and all study groups (except #007-1, which finished before the control group) showed greater reductions compared to the control group (vehicle) (see Table 10 below). In particular, the 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (#003-1 and #004-2) ADC study groups were shown to have significant tumor growth inhibitory effects.

[0306] [Table 10]

[0307] The above results confirm that the ADC group demonstrated statistically significant efficacy compared to the control group.

[0308] Example 4: In vivo efficacy against SCLC in mice (SCLC-H526 cells) Experiments were conducted to evaluate the efficacy of ADC agents against SCLC. The SCLC-H526 (NCI-H526, stage E, carcinoma; mutant small cell lung cancer, c-Kit overexpressing) xenograft model was used.

[0309] The assay was performed on SCLC-H526 cells (2X10 6 The same method as in Example 2 was used, except that a 100 μL / Head / Serum free, RPMI-1640 medium was used. Mice were enrolled in the study 13 days after implantation, and the average tumor volume was approximately 165 mm. 3 (Day 0); imatinib was administered orally for 22 days from D0 to D21. The study groups were #PoC-DM1, #003-1, #004-2 and #003-2.

[0310] The above results are shown in Figures 5a to 5d.

[0311] No abnormal symptoms were observed in any group during the study period prior to sacrifice.

[0312] No weight loss or weight gain of 10% or more was observed for 21 days between the imatinib 100 mg / kg group and the test ADC study groups (1 mg / kg, 3 mg / kg, and 5 mg / kg) compared to the control group (vehicle). Thereafter, no rapid weight loss or rapid weight gain was observed in any of the groups. This suggests that all of the ADC materials of the present invention are very safe.

[0313] On day 21, the imatinib 100mg / kg group showed little tumor growth inhibition compared to the control group (vehicle), whereas the 3 or 5mg / kg ADC study groups showed even stronger tumor growth inhibition effects (with the exception of #007-1 1mg / kg). Notably, and surprisingly, treatment with #004-2 3mg / kg group and #004-2 5mg / kg group induced complete tumor remission without regrowth, even after treatment was discontinued for up to 105 days.

[0314] Tumor weights were measured after sacrifice and all study groups (except #007-1, which was terminated before the control group) showed greater reductions compared to the control group (vehicle) (see Table 11 below). In particular, the 2G4 ThioBridge®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (#004-2) ADC study group was shown to have a significant tumor growth inhibitory effect.

[0315] [Table 11]

[0316] The above results confirm that the ADC group demonstrated statistically significant efficacy compared to the control group.

[0317] Example 5: In vivo efficacy against mast cell tumors in mice (HMC 1.2 cells) An experiment was conducted to evaluate the efficacy of NN3201 (2G4-ThioBridge®-Glu(Val-Cit-PAB-MMAE)-PEG(24u)DAR 4; #004-2) against mast cell tumors. HMC 1.2 (c-Kit mutated and imatinib resistant; Exon11(V560G)+Exon17(D816V)) xenograft model was used.

[0318] Four-week-old female CB-17 SCID immunodeficient mice (Janvier France, Inc.) were used for the experiment after acclimation for 18 days under SPF (specific pathogen free) conditions. Six-week-old mice were subcutaneously injected with HMC-1.2 cells (1X10 6 / 100 μL / Head / Serum free, IMDM medium). The total injection volume containing suspended cells was 200 μL.

[0319] Mice were enrolled in the study 11 days after implantation, with a mean tumor volume of approximately 175.4 mm 3 (Day 0). After random allocation to each group (n=6 / group), mice were administered ADC vehicle (5mL / kg, ivtail), 2G4 (5mg / kg, ivtail), #PoC-DM1 (5mg / kg, ivtail), NN3201 (1, 3 or 5mg / kg, ivtail) and imatinib (100mg / kg, po). The ADC vehicle was a mixture of 20mM histidine (Merck, 104352), 50mM NaCl (Sigma, S3014), 5% sucrose (Sigma, S0389) (pH 6.5) and 0.22μM filter (Merck, S2GPU11RE). Individual doses were calculated based on the animal weight recorded immediately before dosing with a dose volume of 5mL / kg body weight. The iv injections were given three times on D0, D7, and D14, and imatinib was administered orally for 18 days from D0 to D17.

[0320] The results are shown in Figure 6.

[0321] No abnormal symptoms were observed in any group during the study period.

[0322] Compared with the control group (vehicle), no weight loss or weight gain of 10% or more was observed between the imatinib 100 mg / kg group and the test group for 17 days. After that, no rapid weight loss or rapid weight gain was observed in any group. This suggests that all the ADC substances of the present invention are very safe.

[0323] On day 17, the imatinib 100 mg / kg group showed little tumor inhibition, whereas the ADC study group showed a more potent tumor growth inhibitory effect compared to the control group (vehicle).

[0324] Tumors were reinoculated on day 77. On day 95, compared with the control (first) inoculation group, TGI (tumor growth inhibition) was 71.0% in the #NN3201 1 mg / kg (re) inoculation group, 79.0% in the #NN3201 3 mg / kg (re) inoculation group, and 74.8% in the #NN3201 5 mg / kg (re) inoculation group. And the #PoC-DM1 5 mg / kg (re) inoculation group showed 46.2% tumor inhibition (Figure 7).

[0325] The above results confirm that the ADC group demonstrated statistically significant efficacy compared to the control group.

[0326] Example 6: In vivo efficacy against AML in mice (Kasumi-1 cells) This study was conducted to evaluate the efficacy of NN3201 (2G4 ThioBridge®-Glu(Val-Cit-PAB-MMAE)-PEG(24u)DAR 4) against AML. Kasumi-1 (c-Kit mutated and imatinib partial sensitivity; Exon17(N822K)) xenograft model was used.

[0327] The experiment was performed by growing Kasumi-1 cells (3X10 6 The same method as in Example 5 was used, except that a 100 μL / Head / Serum free, RPMI1640 medium was used. Mice were enrolled in the study 29 days after implantation, and the average tumor volume was approximately 170 mm.3 (Day 0).

[0328] The results are shown in Figure 8.

[0329] No abnormal symptoms were observed in any group during the study period.

[0330] During the 17 days, no weight loss or weight gain of 10% or more was observed between the imatinib 100 mg / kg group and the test group compared to the control group (vehicle). After that, no rapid weight loss or rapid weight gain was observed in any group. This suggests that all the ADC materials of the present invention are very safe.

[0331] On day 17, compared with the control group (vehicle), the imatinib 100 mg / kg group showed little inhibition of tumor growth, the PoC-DM1 group showed a weak inhibitory effect, and the NN3201 group showed a stronger tumor growth inhibitory effect.

[0332] The above results confirm that the ADC group demonstrated statistically significant efficacy compared to the control group.

[0333] Example 7: In vivo efficacy against breast cancer in mice (MDA-MB-468 cells) To evaluate the efficacy of ADC agents against breast cancer, we used the MDA-MB-468 (c-Kit, Negative) xenograft model.

[0334] The experiment was performed by growing MDA-MB-468 cells (5X10 6 The same method as in Example 5 was used, except that 100 μL / Head / Serum free, DMEM medium) was used. Mice were enrolled in the study 21 days after implantation, and the average tumor volume was approximately 167 mm. 3 (Day 0).

[0335] The results are shown in Figures 9a to 9d.

[0336] No abnormal symptoms were observed in any group during the study period prior to sacrifice.

[0337] Compared to the control group (vehicle), no weight loss or weight gain of 10% or more was observed between the imatinib 100 mg / kg group and the test ADC study groups (1 mg / kg, 3 mg / kg and 5 mg / kg) for 49 days. After that, no rapid weight loss or rapid weight gain was observed in any group. This suggests that all the ADC materials of the present invention are very safe.

[0338] On day 49, #PoC-DM1 did not show any antitumor activity in xenografts implanted with MDA-MB-468 cells lacking c-Kit expression, compared with the control group (vehicle); on the other hand, #003-1, #004-2 and #003-2 were confirmed to have stronger tumor growth suppression effects at all doses. In particular, the group treated with 5 mg / kg #004-2 was confirmed to significantly suppress tumor growth.

[0339] After sacrifice, tumor weight was measured, and tumor reduction was observed in #003-1, #004-2, and #003-2 compared to the control group (vehicle) (see Table 12 below). In particular, the #004-2 group was shown to have a significant tumor growth inhibitory effect.

[0340] [Table 12]

[0341] An interesting point in the MDA-MB-468 results is that ADCs with 2G4, cleavable linkers and MMAE show dose-dependent efficacy in c-Kit-negative cell-based xenograft models, whereas ADCs with non-cleavable linkers do not, suggesting that ADCs with 2G4, cleavable linkers and MMAE may be ideal partners for non-c-Kit targeting.

[0342] Example 8: In vivo efficacy against SCLC in mice (SCLC-H526 cells) - Rechallenge analysis To determine whether NN3201 treatment was effective following tumor re-inoculation, a tumor re-challenge analysis was performed.

[0343] The analysis was performed in the same manner as in Example 3, except that the experimental groups were 3 mg / kg and 5 mg / kg #004-2 groups alone. Tumor rechallenge was performed at the time of complete tumor remission (day 63). The control cohort (square) used age-matched animals as a control group that received the first tumor inoculation on day 63 without initial tumor implantation on the right flank. For xenograft analysis, SCLC-H526 (2X10 6 / 100 μL / Head / Serum free, RPMI-1640 medium) cells were injected sc (subcutaneously, left flank) into CB-17 SCID mice.

[0344] The results are shown in Figure 10.

[0345] Upon tumor rechallenge, the 3 mg / kg and 5 mg / kg #004-2 groups demonstrated dose-dependent efficacy without additional ADC injections, whereas the control group suffered from tumor growth.

[0346] Example 9: In vivo efficacy against mast cell tumors in mice (HMC 1.2 cells) - Rechallenge analysis To determine whether NN3201 treatment was effective following tumor re-inoculation, a tumor re-challenge analysis was performed.

[0347] Tumor rechallenge analysis was performed in the NN3201 (1, 3 or 5 mg / kg, ivtail) groups at the time point when complete tumor remission was confirmed in Example 4 (day 77). We included the PoC DM1 ADC to understand the difference between DM1 and MMAE as payloads in terms of immunogenic cell death (ICD). Age-matched animals were used as control groups. For xenograft analysis, HMC-1.2 cells (1x10 6 / 100 μL / Head / Serum free, IMDM medium) cells were injected sc (subcutaneously, left flank) into CB-17 SCID mice.

[0348] The results are shown in Figure 11.

[0349] In tumor rechallenge assays, the NN3201 group showed dose-dependent efficacy without further ADC injection, whereas the control group suffered from tumor growth. The PoC-DM1 group showed less tumor growth inhibition than NN3201, indicating that NN3201 had a stronger effect than PoC-DM1.

[0350] Example 10: Preliminary in vivo toxicity analysis (10 mg / kg) An experiment was conducted to evaluate the toxicity of ADC substances by administering a single high-concentration iv dose to Balb / c mice and examining the weight change over a two-week period.

[0351] Seven-week-old female BALB / c mice (Daehan Biolink) were acclimated under SPF (Specific Pathogen Free) conditions before use in the experiments. After randomly allocating to one of six groups (n=3 / group), the mice were administered the ADC vehicle (5mL / kg, ivtail) and each ADC substance (10mg / kg, ivtail). The ADC vehicle buffer was a mixture of 20mM histidine (Merck, 104352), 50mM NaCl (Sigma, S3014), 5% sucrose (Sigma, S0389) (pH 6.5) and 0.22μM filter (Merck, S2GPU11RE). Individual doses were calculated based on the animal weight recorded immediately before administration at a dose volume of 5mL / kg body weight (Table 13).

[0352] [Table 13]

[0353] All animals were observed once a day for mortality, general condition and clinical signs (time, onset, severity and recovery). All animals were weighed daily from the day of grouping. The animals were sacrificed on the 14th day.

[0354] After the experiment, the animals were euthanized (cervical dislocation was performed as quickly as possible to minimize pain, since pulmonary congestion may be affected during euthanasia using respiratory anesthetics or a CO2 chamber), and a necropsy examination was performed to confirm the presence or absence of pulmonary congestion symptoms. The tissues (lungs) were preserved in 10% NBF (neutral buffered formalin).

[0355] The results are shown in Figure 12.

[0356] No abnormal symptoms were observed in any group during the study period.

[0357] During the study period, no symptoms related to weight gain or loss (5% or more) due to administration were observed in any group.

[0358] At the end of the experiment (Day 14), the body weight increased by 0.43g (2.10%) in the #003-1 10mg / kg group and by 0.63g (3.00%) in the #004-2 10mg / kg group compared to the day of administration (Day 0). The body weight of the #003-2 10mg / kg group decreased by 0.27g (1.29%) at the end of the experiment (Day 14) compared to the day of administration (Day 0).

[0359] There were no signs of pulmonary congestion in lung tissues from any group.

[0360] Example 11: Preliminary in vivo toxicity analysis (20 / 40 / 60 mg / kg) An experiment was conducted to evaluate the toxicity of ADC substances by administering a single high-concentration iv dose to Balb / c mice and examining the weight change over a two-week period.

[0361] The analysis was carried out in the same manner as in Example 10, except that the groups were distributed as follows (Table 14):

[0362] [Table 14]

[0363] The results are shown in Figures 13a to 13d.

[0364] No abnormal symptoms were observed in any group during the study period.

[0365] During the study period, no symptoms of weight loss or weight gain (>5%) due to administration were observed in any group. On the first day after administration (Day 0), weight loss of 0.63 g (3.23%) was observed in the #PoC-DM1 40 mg / kg group and 0.70 g (3.57%) in the #PoC-DM1 60 mg / kg group. However, the lost weight was recovered.

[0366] Although the embodiments of the present invention have been described above, a person having ordinary knowledge in the art may modify and change the present invention in various ways by adding, changing, deleting or adding components, etc., without departing from the spirit of the present invention as described in the claims, and this can also be said to be included in the scope of the present invention.

Claims

1. An antibody-drug conjugate of the following formula 1: <Chemical formula 1> A-(L) ｘ -(D) ｙ In the above formula, Ab is an anti-c-Kit antibody or antigen-binding fragment thereof that specifically binds to the epitopes of human c-Kit in SEQ ID NO:9 and SEQ ID NO:10; L is a linker, including a cleavable linker; D is a drug moiety; x is an integer from 1 to 8; and y is an integer from 1 to 8.

2. The antibody-drug conjugate of claim 1, wherein the Ab is an antibody or an antigen-binding fragment thereof that specifically binds to one or more of R122, Y125, R181, K203, R205, S261 and H263 in SEQ ID NO:

12.

3. 2. The antibody-drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof cross-competes with a reference antibody comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6 for binding to epitopes of human c-Kit represented by SEQ ID NOs: 9 and 10.

4. The antibody-drug conjugate of claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5 and a light chain CDR3 of SEQ ID NO:

6.

5. The antibody-drug conjugate of claim 1 , wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain represented by SEQ ID NO: 7 and a light chain variable domain represented by SEQ ID NO:

8.

6. The antibody-drug conjugate of claim 3 or 5, wherein the antibody or antigen-binding fragment thereof has one or two amino acids in the CDR modified, deleted or substituted.

7. The antibody-drug conjugate of any one of claims 1 to 6, characterized in that the antibody or antigen-binding fragment thereof maintains greater than 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity across the heavy chain variable or light chain heavy domains.

8. The antibody-drug conjugate of claim 1, wherein L is a linker containing a spacer in a form in which a polymer is bound or not bound.

9. 2. The antibody-drug conjugate of claim 1, wherein the linker L has the structure of Formula 2: <Chemical formula 2> (L １ ) ｍ -(S) ｎ -(L ２ ) ｐ In the above formula, L １ is the Ab and S or Ab and L ２ is a linker linking S is a spacer in either polymer-attached or unattached form; L ２ is a cleavable linker; m is an integer from 0 to 8; n is an integer from 0 to 8; p is an integer from 1 to 8.

10. The antibody-drug conjugate according to claim 9, wherein m is 1 to 4, n is 1 to 4, and p is 1 to 4.

11. Said L １ The antibody-drug conjugate of claim 9, further comprising a linker selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a procharged linker, and a dicarboxylic acid-based linker.

12. The L or L １ teeth, The antibody-drug conjugate according to claim 1 or 9,

13. The S is The antibody-drug conjugate of claim 9, further comprising a spacer selected from the group consisting of:

14. The antibody-drug conjugate of claim 9, wherein S is a spacer comprising an aspartate, a glutamate, or a combination thereof, with or without a polymer attached thereto.

15. 10. The antibody-drug conjugate of claim 9, wherein the polymer is polyalkylene, polyalkylene glycol, polyvinylpyrrolidone, polyacrylate, polyoxazoline, polyvinylalcohol, polyacrylamide or polymethacrylamide, HPMA copolymer, polyester, polyacetal, poly(orthoester), polycarbonate, poly(iminocarbonate), polyamide, divinylether-maleic anhydride or styrene-maleic anhydride copolymer, polysaccharide, or polyglutamic acid.

16. Said L ２ 10. The antibody-drug conjugate of claim 9, comprising a linker selected from the group consisting of valine-citrulline-p-aminobenzylcarbamoyl (Val-Cit-PAB), alanine-phenylalanine-p-aminobenzylcarbamoyl (Ala-Phe-PAB), alanine-alanine-p-aminobenzylcarbamoyl (Ala-Ala-PAB), valine-alanine-p-aminobenzylcarbamoyl (Val-Ala-PAB), phenylalanine-lysine-p-aminobenzylcarbamoyl (Phe-Lys-PAB), alanine-alanine-glycine-p-aminobenzylcarbamoyl (Ala-Ala-Gly-PAB), and glycine-glycine-glycine-p-aminobenzylcarbamoyl (Gly-Gly-Gly-PAB) linkers.

17. The antibody-drug conjugate of claim 1, wherein the linker (L) comprises a linker selected from the group consisting of the following formulas 3 to 5: <Chemical formula 3> <Chemical formula 4> <Chemical formula 5>

18. 2. The antibody-drug conjugate of claim 1, wherein the drug moiety (D) is selected from the group consisting of V-ATPase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule inhibitors, auristatins, dolastatins, maytansinoids, MetAP (methionine aminopeptidase), inhibitors of nuclear export of protein CRM1, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binding agents and DHFR inhibitors.

19. 20. The antibody-drug conjugate of claim 18, wherein the drug moiety (D) is a microtubule inhibitor (MTI) moiety.

20. 20. The antibody-drug conjugate of claim 19, wherein the drug moiety (D) comprises a drug selected from the group consisting of SN-38 ((4S)-4,11-Diethyl-4,9-dihydroxy-1,4-dihydro-3H,14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione), auristatin, dolastatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), monomethydolastatin 10 (MMAD), or a combination thereof.

21. The antibody-drug conjugate of claim 1, wherein the antibody-drug conjugate is selected from the group consisting of the following formulas 6 to 8: <Chemical formula 6> <Chemical formula 7> <Chemical formula 8> In the above formula, a is an integer from 1 to 8, and b and c are each an integer from 1 to 4.

22. An antibody-drug conjugate selected from the group consisting of the following formulas 6 to 8: <Chemical formula 6> <Chemical formula 7> <Chemical formula 8> In the above formula, Ab is an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 of SEQ ID NO:1, a heavy chain CDR2 of SEQ ID NO:2, a heavy chain CDR3 of SEQ ID NO:3, a light chain CDR1 of SEQ ID NO:4, a light chain CDR2 of SEQ ID NO:5, and a light chain CDR3 of SEQ ID NO:6; a is an integer from 1 to 8, and b and c are each an integer from 1 to 4.

23. A pharmaceutical composition for preventing or treating cancer, comprising the antibody-drug conjugate of claim 1 or 22.

24. 27. A method for preventing or treating cancer, comprising administering to a subject in need thereof a pharma- ceutical effective amount of a composition comprising the antibody-drug conjugate of claim 1 or 22.

25. The antibody-drug conjugate of claim 1 for use in therapy.

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